Identification of variable influenza residues and uses thereof

ABSTRACT

Provided herein are universal prophylactic compositions for preventing infection with influenza viruses by directing the immune response to highly conserved regions of the virus. Also provided are universal therapeutic compositions for treating influenza infection by targeting the highly conserved regions. Methods for using the prophylactic and therapeutic compositions are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/983,519, filed Feb. 28, 2020, the entirecontents of which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.FA8702-15-D-0001 awarded by the U.S. Air Force. The Government hascertain rights in the invention.

SEQUENCE LISTING STATEMENT

A computer readable form of the Sequence Listing is filed with thisapplication by electronic submission and is incorporated into thisapplication by reference in its entirety. The Sequence Listing iscontained in the file created on Aug. 18, 2022 having the file name“21-0895-US_SeqList2.txt” and is 170 in size.

BACKGROUND

Influenza viruses are members of the family Orthomyxoviridae and aredivided into three genera: A, B, and C. Influenza A and B viruses causerespiratory infections in humans. Current vaccines are designed toinduce immunity to hemagglutinin, one of two glycoproteins present onthe surface of influenza viruses. Despite the availability of highlyeffective vaccines, influenza infection still results in up to 5,000,000hospitalizations and 500,000 deaths annually worldwide. Currentlyavailable vaccines against influenza include up to four influenzahemagglutinin components intended to provide protection against H1N1,H3N2, and influenza B strains. Vaccine compositions are reassessedannually by the World Health Organization (WHO) to accommodate antigenicshift and drift in circulating virus strains. Such a strategy requiresdiligent surveillance of circulating influenza strains from year toyear, and vaccine mismatches resulting from inaccurate predictions orunpredictable HA mutations arising during vaccine manufacture, which canresult in increased morbidity and mortality even in vaccinatedpopulations.

Given the shortcomings of the currently available vaccines, thereremains a need for prophylactic and therapeutic compositions and methodsthat can be used to broadly target influenza in view of the high virusmutation rate amongst strains.

SUMMARY OF THE INVENTION

The present disclosure provides immunogenic compositions, methods forimmunizing a subject against infection with an influenza virus, methodsfor inducing an immune response against influenza virus, and methods ofreducing an influenza virus infection in a subject in need thereof byadministering one or more immunogenic compositions of the disclosure.

In one aspect, the disclosure provides an immunogenic compositioncomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more hypervariable amino acidresidues and one or more conserved amino acid residues, wherein theamino acid sequence of the polypeptide comprises a substitution of atleast one hypervariable amino acid residue with a different,non-hypervariable amino acid residue. In one embodiment, the at leastone non-hypervariable amino acid residue is a nonpolar, aliphatic Rgroup amino acid selected from alanine, glycine, valine, leucine,isoleucine, and methionine. In one embodiment, the non-hypervariableamino acid residue is alanine or glycine.

In one aspect, the disclosure provides an immunogenic compositioncomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more hypervariable amino acidresidues and one or more conserved amino acid residues, wherein theamino acid sequence of the polypeptide comprises a substitution of atleast one hypervariable amino acid residue with an amino acid residuethat is a hypervariable-substitute. In one embodiment, thehypervariable-substitute is a nonpolar, aliphatic R group amino acidselected from alanine, glycine, valine, leucine, isoleucine, andmethionine. In one embodiment, the hypervariable-substitute is alanineor glycine.

In one aspect, the disclosure provides an immunogenic compositioncomprising two or more polypeptides each individually comprising anamino acid sequence of a viral protein comprising one or morehypervariable amino acid residues, wherein each polypeptide individuallycomprises an amino acid sequence having a substitution of at least onehypervariable amino acid residue with a different, non-hypervariableamino acid residue, and wherein the polypeptides are of the same ordifferent influenza virus strains. In one embodiment, the at least onenon-hypervariable amino acid residue is a nonpolar, aliphatic R groupamino acid selected from alanine, glycine, valine, leucine, isoleucine,and methionine. In one embodiment, the non-hypervariable amino acidresidue is alanine or glycine.

In one aspect, the disclosure provides an immunogenic compositioncomprising two or more polypeptides each individually comprising anamino acid sequence of a viral protein comprising one or morehypervariable amino acid residues, wherein each polypeptide individuallycomprises an amino acid sequence having a substitution of at least onehypervariable amino acid residue with an amino acid residue that is ahypervariable-substitute, and wherein the polypeptides are of the sameor different influenza virus strains. In one embodiment, thehypervariable-substitute is a nonpolar, aliphatic R group amino acidselected from alanine, glycine, valine, leucine, isoleucine, andmethionine. In one embodiment, the hypervariable-substitute is alanineor glycine.

In one aspect, the disclosure provides an immunogenic compositioncomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more hypervariable amino acidresidues and one or more conserved amino acid residues, wherein theinfluenza virus is an influenza A virus strain or an influenza B virusstrain, wherein the amino acid sequence of the polypeptide comprises asubstitution of at least one hypervariable amino acid residue with adifferent, non-hypervariable amino acid residue. In one embodiment, theat least one non-hypervariable amino acid residue is a nonpolar,aliphatic R group amino acid selected from alanine, glycine, valine,leucine, isoleucine, and methionine. In one embodiment, thenon-hypervariable amino acid residue is alanine or glycine.

In one aspect, the disclosure provides an immunogenic compositioncomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more hypervariable amino acidresidues and one or more conserved amino acid residues, wherein theinfluenza virus is an influenza A virus strain or an influenza B virusstrain, wherein the amino acid sequence of the polypeptide comprises asubstitution of at least one hypervariable amino acid residue with anamino acid residue that is a hypervariable-substitute. In oneembodiment, the hypervariable substitute is a nonpolar, aliphatic Rgroup amino acid selected from alanine, glycine, valine, leucine,isoleucine, and methionine. In one embodiment, thehypervariable-substitute is alanine or glycine.

In one aspect, the disclosure provides an immunogenic compositioncomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more hypervariable amino acidresidues and one or more conserved amino acid residues, wherein thevirus influenza virus is H1N1, H3N2, B/Victoria/2/1987-like,B/Yamagata/16/1988-like, H5N1, or any combination thereof, wherein theamino acid sequence of the polypeptide comprises a substitution of atleast one hypervariable amino acid residue with a different,non-hypervariable amino acid residue. In one embodiment, the at leastone non-hypervariable amino acid residue is a nonpolar, aliphatic Rgroup amino acid selected from alanine, glycine, valine, leucine,isoleucine, and methionine. In one embodiment, the non-hypervariableamino acid residue is alanine or glycine.

In one aspect, the disclosure provides an immunogenic compositioncomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more hypervariable amino acidresidues and one or more conserved amino acid residues, wherein thevirus influenza virus is H1N1, H3N2, B/Victoria/2/1987-like,B/Yamagata/16/1988-like, H5N1, or any combination thereof, wherein theamino acid sequence of the polypeptide comprises a substitution of atleast one hypervariable amino acid residue with an amino acid residuethat is a hypervariable-substitute. In one embodiment, the hypervariablesubstitute is a nonpolar, aliphatic R group amino acid selected fromalanine, glycine, valine, leucine, isoleucine, and methionine. In oneembodiment, the hypervariable-substitute is alanine or glycine.

In one aspect, the disclosure provides an immunogenic compositioncomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more hypervariable amino acidresidues and one or more conserved amino acid residues, wherein the atleast one viral protein is a hemagglutinin protein, a neuraminidaseprotein, a M2 matrix protein, or combinations thereof, wherein the aminoacid sequence of the polypeptide comprises a substitution of at leastone hypervariable amino acid residue with a different, non-hypervariableamino acid residue. In one embodiment, the at least onenon-hypervariable amino acid residue is a nonpolar, aliphatic R groupamino acid selected from alanine, glycine, valine, leucine, isoleucine,and methionine. In one embodiment, the non-hypervariable amino acidresidue is alanine or glycine.

In one aspect, the disclosure provides an immunogenic compositioncomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more hypervariable amino acidresidues and one or more conserved amino acid residues, wherein the atleast one viral protein is a hemagglutinin protein, a neuraminidaseprotein, a M2 matrix protein, or combinations thereof, wherein the aminoacid sequence of the polypeptide comprises a substitution of at leastone hypervariable amino acid residue with an amino acid that is ahypervariable-substitute. In one embodiment, the hypervariablesubstitute is a nonpolar, aliphatic R group amino acid selected fromalanine, glycine, valine, leucine, isoleucine, and methionine. In oneembodiment, the hypervariable-substitute is alanine or glycine.

In one aspect, the disclosure provides an immunogenic compositioncomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more hypervariable amino acidresidues and one or more conserved amino acid residues, wherein theamino acid sequence of the polypeptide comprises a substitution of atleast one hypervariable amino acid residue with a different,non-hypervariable amino acid residue, and wherein the polypeptidecomprises at least one B cell epitope. In one embodiment, the at leastone non-hypervariable amino acid residue is a nonpolar, aliphatic Rgroup amino acid selected from alanine, glycine, valine, leucine,isoleucine, and methionine. In one embodiment, the non-hypervariableamino acid residue is alanine or glycine. In one embodiment, theimmunogenic composition elicits an immune response against the at leastone B cell epitope. In one embodiment, the immune response comprisesproduction of antibodies that bind the at least one B cell epitope.

In one aspect, the disclosure provides an immunogenic compositioncomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more hypervariable amino acidresidues and one or more conserved amino acid residues, wherein theamino acid sequence of the polypeptide comprises a substitution of atleast one hypervariable amino acid residue with an amino acid that is ahypervariable-substitute, and wherein the polypeptide comprises at leastone B cell epitope. In one embodiment, the hypervariable substitute is anonpolar, aliphatic R group amino acid selected from alanine, glycine,valine, leucine, isoleucine, and methionine. In one embodiment, thehypervariable-substitute is alanine or glycine. In one embodiment, theimmunogenic composition elicits an immune response against the at leastone B cell epitope. In one embodiment, the immune response comprisesproduction of antibodies that bind the at least one B cell epitope.

In one aspect, the disclosure provides an immunogenic compositioncomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more hypervariable amino acidresidues and one or more conserved amino acid residues, wherein theviral protein comprises an amino acid sequence selected from SEQ ID NOs:1-6, and wherein the amino acid sequence of the polypeptide comprises asubstitution of at least one hypervariable amino acid residue with adifferent, non-hypervariable amino acid residue. In one embodiment, thepolypeptide comprises at least one B cell epitope. In one embodiment,the at least one non-hypervariable amino acid residue is a nonpolar,aliphatic R group amino acid selected from alanine, glycine, valine,leucine, isoleucine, and methionine. In one embodiment, thenon-hypervariable amino acid residue is alanine or glycine. In oneembodiment, the immunogenic composition elicits an immune responseagainst the at least one B cell epitope. In one embodiment, the immuneresponse comprises production of antibodies that bind the at least one Bcell epitope. In one embodiment, the hypervariable amino acid which issubstituted is selected from one or more underlined amino acid residuesset forth in SEQ ID NOs: 1-6.

In one aspect, the disclosure provides an immunogenic compositioncomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more hypervariable amino acidresidues and one or more conserved amino acid residues, wherein theviral protein comprises an amino acid sequence selected from SEQ ID NOs:1-6, and wherein the amino acid sequence of the polypeptide comprises asubstitution of at least one hypervariable amino acid residue with anamino acid that is a hypervariable-substitute. In one embodiment, thepolypeptide comprises at least one B cell epitope. In one embodiment,the at least one non-hypervariable amino acid residue is a nonpolar,aliphatic R group amino acid selected from alanine, glycine, valine,leucine, isoleucine, and methionine. In one embodiment, thenon-hypervariable amino acid residue is alanine or glycine. In oneembodiment, the immunogenic composition elicits an immune responseagainst the at least one B cell epitope. In one embodiment, the immuneresponse comprises production of antibodies that bind the at least one Bcell epitope. In one embodiment, the hypervariable amino acid which issubstituted is selected from one or more underlined amino acid residuesset forth in SEQ ID NOs: 1-6.

In any of the foregoing or related embodiments, the immunogeniccomposition further comprises an adjuvant.

In any of the foregoing and related aspects, the immunogenic compositioncomprises a nucleic acid encoding the at least one polypeptide.

In one aspect, the disclosure provides a method for immunizing a subjectagainst infection with an influenza virus, comprising administering oneor more immunogenic compositions of the disclosure.

In one aspect, the disclosure provides a method for inducing an immuneresponse against influenza virus, comprising administering to a subjectone or more immunogenic compositions of the disclosure.

In one aspect, the disclosure provides a method of reducing an influenzavirus infection in a subject in need thereof, comprising administeringto a subject one or more immunogenic compositions of the disclosure.

In any of the foregoing and related aspects, the administration of oneor more immunogenic compositions to the subject results in theproduction of antibodies against the at least one B cell epitope in thepolypeptide.

Other aspects of the disclosure relate to methods for generating animmunogenic composition comprising:

(i) obtaining two or more amino acid sequences of viral proteins fromone or more strains of a particular type and/or subtype of influenzavirus;

(ii) aligning the amino acid sequences to generate an alignment;

(iii) identifying one or more hypervariable amino acid residues betweenstrains and one or more conserved amino acid residues; and

(iv) substituting at least one hypervariable amino acid residueidentified in (iii) with a different, non-hypervariable amino acidresidue. In some aspects, the alignment is generated with Dawn, orClustal-Omega. In some aspects, the method further comprises performingsite-specific mutagenesis at each hypervariable amino residue, orcombinations thereof, and determining if the mutated viral proteinelicits neutralizing antibodies against the multiple strains ofinfluenza virus.

Other aspects of the disclosure relate to immunogenic compositionscomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more amino acid residues whichare conserved between one or more strains of a type and/or subtype ofinfluenza virus, wherein the amino acid sequence of the polypeptidecomprises an amino acid sequence comprising the one or more conservedamino acid residues.

In one aspect, the disclosure provides an immunogenic compositioncomprising two or more polypeptides, each individually comprising anamino acid sequence of a viral protein having one or more amino acidresidues which are conserved between one or more strains of a typeand/or subtype of influenza virus, wherein each polypeptide individuallycomprises an amino acid sequence comprising one or more conserved aminoacid sequences, and wherein the polypeptides are of the same ordifferent influenza virus strains. In some aspects, the two or morepolypeptides are of the same viral protein. In some aspects the two ormore polypeptides are of different viral proteins.

In one aspect the disclosure relates to immunogenic compositionscomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more amino acid residues whichare conserved between one or more strains of a type and/or subtype ofinfluenza virus, wherein the amino acid sequence of the polypeptidecomprises an amino acid sequence comprising the one or more conservedamino acid residues, and wherein the polypeptide comprises two or more Tcell epitopes, wherein each T cell epitope is operably linked to oneother, optionally via a linker.

In one aspect, the disclosure provides immunogenic compositionscomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more amino acid residues whichare conserved between one or more strains of a type and/or subtype ofinfluenza virus, wherein the influenza virus is an influenza A virusstrain or an influenza B virus strain, and wherein the amino acidsequence of the polypeptide comprises an amino acid sequence comprisingthe one or more conserved amino acid residues.

In one aspect, the disclosure provides immunogenic compositionscomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more amino acid residues whichare conserved between one or more strains of a type and/or subtype ofinfluenza virus, wherein the influenza virus is H1N1, H3N2,B/Victoria/2/1987-like, B/Yamagata/16/1988-like, H5N1, or anycombination thereof, and wherein the amino acid sequence of thepolypeptide comprises an amino acid sequence comprising the one or moreconserved amino acid residues.

In one aspect, the disclosure provides immunogenic compositionscomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more amino acid residues whichare conserved between one or more strains of a type and/or subtype ofinfluenza virus, wherein the viral protein is a hemagglutinin protein, aneuraminidase protein, a M2 matrix protein, or combinations thereof, andwherein the amino acid sequence of the polypeptide comprises an aminoacid sequence comprising the one or more conserved amino acid residues.

In one aspect, the disclosure provides immunogenic compositionscomprising at least one polypeptide comprising an amino acid sequence ofan influenza viral protein having one or more amino acid residues whichare conserved between one or more strains of a type and/or subtype ofinfluenza virus, wherein the at least one viral polypeptide comprises atleast one conserved amino acid sequence selected from SEQ ID NOs: 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 151, 153 and 155, and any combination thereof, andwherein the amino acid sequence of the polypeptide comprises an aminoacid sequence comprising the one or more conserved amino acid residues.

In any of the foregoing and related aspects, the immunogenic compositioncomprises a nucleic acid encoding the at least one polypeptide.

In any of the foregoing and related aspects, the immunogenic compositionelicits an immune response against the virus. In some aspects, theimmune response is a T cell response directed to the one of more T cellepitopes comprising the conserved amino acid residues of the viralprotein.

In any of the foregoing and related aspects, the immunogenic compositionfurther comprises an adjuvant.

In some aspects the disclosure provides methods of immunizing a subjectagainst infection with an influenza virus, optionally a T cell or B cellresponse or both, comprising administering one or more immunogeniccompositions of the disclosure.

In some aspects the disclosure provides methods for inducing an immuneresponse against influenza virus, optionally a T cell or B cell responseor both, comprising administering one or more immunogenic compositionsof the disclosure.

In some aspects the disclosure provides methods of reducing an influenzavirus infection in a subject in need thereof, optionally a T cell or Bcell response or both, comprising administering one or more immunogeniccompositions of the disclosure.

In some aspects, the composition elicits a T cell response against oneor more T cell epitopes comprising the conserved amino acid residues ofthe viral protein.

In other aspects, the disclosure provides methods for generating animmunogenic composition comprising:

(i) obtaining two or more amino acid sequences of viral proteins frommultiple strains of a particular type and/or subtype of influenza virus;

(ii) aligning the amino acid sequences to generate an alignment;

(iii) identifying a region of amino acid residues having conserved aminoacid residues between strains; and

(iv) generating a polypeptide comprising the region of amino acidsidentified in (iii). In one embodiment, the alignment is generated withDawn or Clustal-Omega.

In one embodiment, the methods of the disclosure further comprisedetermining if the immunogenic composition elicits a T cell responseagainst the multiple strains of influenza virus.

In some aspects, the disclosure provides an immunogenic compositioncomprising:

(a) one or more immunogenic compositions comprising at least onepolypeptide comprising an amino acid sequence of an influenza viralprotein having one or more hypervariable amino acid residues and one ormore conserved amino acid residues, optionally wherein the influenzavirus is an influenza A virus strain or an influenza B virus strain, andwherein the amino acid sequence of the polypeptide comprises asubstitution of at least one hypervariable amino acid residue with adifferent, non-hypervariable amino acid residue; and

(b) one or more immunogenic compositions comprising at least onepolypeptide comprising an amino acid sequence of an influenza viralprotein having one or more amino acid residues which are conservedbetween one or more strains of a type and/or subtype of influenza virus,optionally wherein the influenza virus is an influenza A virus strain oran influenza B virus strain, and wherein the amino acid sequence of thepolypeptide comprises an amino acid sequence comprising the one or moreconserved amino acid residues. In one embodiment, the at least onenon-hypervariable amino acid residue in an immunogenic composition (a)is a nonpolar, aliphatic R group amino acid selected from alanine,glycine, valine, leucine, isoleucine, and methionine. In one embodiment,the non-hypervariable amino acid residue is alanine or glycine.

In one embodiment, the at least one polypeptide of (a) and the at leastone polypeptide of (b) are from the same or different viral proteinsfrom the same influenza virus type. In one embodiment, the at least onepolypeptide of (a) and the at least on polypeptide of (b) are from thesame or different proteins from different influenza virus types. In oneembodiment, the at least one polypeptide of (b) comprises two or morepolypeptides each individually comprising a T cell epitope. In oneembodiment, the two or more polypeptides comprise same amino acidsequence. In one embodiment, the two or more polypeptides comprisedifferent amino acid sequences. In one embodiment, the two or morepolypeptides are derived from the same viral protein. In one embodimentthe two or more polypeptides are derived from different viral proteins.In one embodiment, the influenza virus is H1N1, H3N2,B/Victoria/2/1987-like, B/Yamagata/16/1988-like, H5N1, or anycombination thereof. In one embodiment, the two or more polypeptides areoperably linked to each other, optionally comprising a linker and/orspacer between each polypeptide. In one embodiment, the one or morecompositions of (a) and the one ore more compositions of (b) furthercomprise an adjuvant.

In some aspects, the disclosure provides an immunogenic compositioncomprising:

(a) one or more immunogenic compositions comprising at least onepolypeptide comprising an amino acid sequence of an influenza viralprotein having one or more hypervariable amino acid residues and one ormore conserved amino acid residues, the viral protein is a hemagglutininprotein, a neuraminidase protein, a M2 matrix protein, or combinationsthereof, and wherein the amino acid sequence of the polypeptidecomprises a substitution of at least one hypervariable amino acidresidue with a different, non-hypervariable amino acid residue; and

(b) one or more immunogenic compositions comprising at least onepolypeptide comprising an amino acid sequence of an influenza viralprotein having one or more amino acid residues which are conservedbetween one or more strains of a type and/or subtype of influenza virus,the viral protein is a hemagglutinin protein, a neuraminidase protein, aM2 matrix protein, or combinations thereof, and wherein the amino acidsequence of the polypeptide comprises an amino acid sequence comprisingthe one or more conserved amino acid residues. In one embodiment, the atleast one non-hypervariable amino acid residue in an immunogeniccomposition (a) is a nonpolar, aliphatic R group amino acid selectedfrom alanine, glycine, valine, leucine, isoleucine, and methionine. Inone embodiment, the non-hypervariable amino acid residue is alanine orglycine. In one embodiment, the immunogenic composition elicits animmune response against at least one T cell epitope, at least one B cellepitope, or combinations thereof. In one embodiment, the immune responsecomprises production of antibodies that bind B cell epitopes, elicitinga T cell response against T cell epitopes, or both.

In some aspects, the disclosure provides an immunogenic compositioncomprising:

(a) one or more immunogenic compositions comprising at least onepolypeptide comprising an amino acid sequence of an influenza viralprotein having one or more hypervariable amino acid residues and one ormore conserved amino acid residues, wherein the at least one polypeptidecomprises an amino acid sequence selected from SEQ ID NOs: 1-6, orcombinations thereof, and wherein the amino acid sequence of thepolypeptide comprises a substitution of at least one hypervariable aminoacid residue with a different, non-hypervariable amino acid residue; and

(b) one or more immunogenic compositions comprising at least onepolypeptide comprising an amino acid sequence of an influenza viralprotein having one or more amino acid residues which are conservedbetween one or more strains of a type and/or subtype of influenza virus,and wherein the amino acid sequence of the polypeptide comprises anamino acid sequence comprising the one or more conserved amino acidresidues. In one embodiment, the at least one non-hypervariable aminoacid residue in an immunogenic composition (a) is a nonpolar, aliphaticR group amino acid selected from alanine, glycine, valine, leucine,isoleucine, and methionine. In one embodiment, the non-hypervariableamino acid residue is alanine or glycine. In one embodiment, theimmunogenic composition elicits an immune response against at least oneT cell epitope, at least one B cell epitope, or combinations thereof. Inone embodiment, the immune response comprises production of antibodiesthat bind B cell epitopes, eliciting a T cell response against T cellepitopes, or both.

In some aspects, the disclosure provides an immunogenic compositioncomprising:

(a) one or more immunogenic compositions comprising at least onepolypeptide comprising an amino acid sequence of an influenza viralprotein having one or more hypervariable amino acid residues and one ormore conserved amino acid residues, wherein the amino acid sequence ofthe polypeptide comprises a substitution of at least one hypervariableamino acid residue with a different, non-hypervariable amino acidresidue, and wherein the hypervariable amino acid which is substitutedis selected from one or more underlined amino acid residues set forth inSEQ ID NOs: 1-6; and

(b) one or more immunogenic compositions comprising at least onepolypeptide comprising an amino acid sequence of an influenza viralprotein having one or more amino acid residues which are conservedbetween one or more strains of a type and/or subtype of influenza virus,and wherein the amino acid sequence of the polypeptide comprises anamino acid sequence comprising the one or more conserved amino acidresidues. In one embodiment, the at least one non-hypervariable aminoacid residue in an immunogenic composition (a) is a nonpolar, aliphaticR group amino acid selected from alanine, glycine, valine, leucine,isoleucine, and methionine. In one embodiment, the non-hypervariableamino acid residue is alanine or glycine. In one embodiment, theimmunogenic composition elicits an immune response against at least oneT cell epitope, at least one B cell epitope, or combinations thereof. Inone embodiment, the immune response comprises production of antibodiesthat bind B cell epitopes, eliciting a T cell response against T cellepitopes, or both.

In one embodiment, the at least one polypeptide of (b) comprises atleast one conserved amino acid sequence selected from SEQ ID NOs: 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 151, 153, and 155, or any combination thereof.

In any of the foregoing or related aspects, the immunogenic compositioncomprises a nucleic acid encoding the at least one polypeptide of (b).

Other aspects of the disclosure relate to methods for immunizing asubject against infection with an influenza virus, comprisingadministering one or more immunogenic composition of (a) and one or moreimmunogenic compositions of (b).

Other aspects of the disclosure relate to methods for inducing an immuneresponse in a subject to protect against infection with an influenzavirus, or reducing an influenza virus infection comprising administeringone or more immunogenic composition of (a) and one or more immunogeniccompositions of (b).

In one embodiment, the immunogenic composition of (a) is administeredprior to, simultaneously with, or subsequently to administration of theimmunogenic composition (b).

Other aspects of the disclosure relate to methods for inducing an immuneresponse or reducing an influenza virus infection in a subject in needthereof who has received or is receiving one or more compositions of(a), the method comprising: administering to the subject an effectiveamount of one or more compositions of (b).

Other aspects of the disclosure relate to methods for inducing an immuneresponse in a subject in need thereof who has received or is receivingone or more compositions of (b), the method comprising: administering tothe subject an effective amount of one or more compositions of (a).

Other aspects of the disclosure relate to methods for reducing aninfluenza virus infection in a subject, comprising administering to thesubject an immunogenic composition comprising: one or more compositionsof any of (a); and one or more compositions of (b). In one embodiment,the immunogenic composition of (a) is administered prior to,simultaneously with, or subsequently to administration of theimmunogenic composition (b).

In other aspects, the disclosure provides nucleic acid moleculescomprising a nucleotide sequence having substantial complementarity to anucleotide sequence encoding a polypeptide derived from a viral proteinof influenza, wherein the at least one polypeptide comprises conservedamino acid sequence between multiple strains of a particular type and/orsubtype of influenza virus. In some embodiments, the nucleic acidmolecule is an RNA interference (RNAi) molecule. In some embodiments,the RNAi molecule is an siRNA or miRNA molecule. In some embodiments,the nucleic acid molecule is an antisense oligonucleotide. In someembodiments, the nucleic acid encodes for one or more polypeptides. Insome embodiment, the one or more polypeptides comprise the same aminoacid sequence. In some embodiments, the one or more polypeptidescomprise different amino acid sequences. In some embodiments, whereinthe one or more polypeptides are derived from the same viral protein. Insome embodiments, wherein the one or more polypeptides are derived fromdifferent viral proteins. In some embodiments, the one or morepolypeptides are operably linked to each other, optionally comprising alinker and/or spacer between each polypeptide. In some embodiments, thenucleic acid is formulated in a composition comprising an adjuvant. Insome embodiments, the influenza virus is an influenza A virus strain oran influenza B virus strain. In some embodiments, the influenza virus isH1N1, H3N2, B/Victoria/2/1987-like, B/Yamagata/16/1988-like, H5N1, orany combination thereof. In some embodiments, the viral protein is ahemagglutinin protein, a neuraminidase protein, a M2 matrix protein, orcombinations thereof. In some embodiments, the composition elicits animmune response against the virus. In some embodiments, the immuneresponse is a T cell response directed to one of more T cell epitopes.In some embodiments, the nucleic acid encodes a conserved amino acidsequence selected from SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 151, 153 and 155,or any combination thereof.

In some embodiments, the disclosure provides method for immunizing asubject against infection with an influenza virus, methods for inducingan immune response against influenza virus, and methods of reducing aninfluenza virus infection in a subject in need thereof, comprisingadministering the nucleic acid molecule of the disclosure. In someembodiments, the nucleic acid molecule elicits a T cell responsedirected to one of more T cell epitopes. In some embodiments, the methodfurther comprises determining if the nucleic acid molecule elicits a Tcell response against the multiple strains of influenza virus.

In other aspects, the disclosure provides methods of treating aninfluenza infection, comprising administering the nucleic acid moleculeof the disclosure, optionally in a delivery vehicle.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a schematic showing yearly variations in hemagglutinin aminoacid sequence from influenza A H1N1.

FIG. 2 provides the amino acid sequence (SEQ ID NO: 1) and nucleic acidsequence (SEQ ID NO: 169) of influenza A hemagglutinin. Hypervariableresidues are indicated by a box whereas highly conserved regions areunderlined.

FIG. 3 provides the amino acid sequence (SEQ ID NO: 157) and nucleicacid sequence (SEQ ID NO: 170) of influenza A hemagglutinin wherehypervariable residues have been replaced with alanine (shown using abox).

DETAILED DESCRIPTION

The present disclosure provides compositions and methods for inducing animmune response across strains of influenza virus. Specifically,hypervariable residues and highly conserved regions of amino acidsequences have been identified in various influenza viral proteins thatcan be exploited to induce a universal immune response amongst strains.

Identification of Influenza Residues

Influenza is caused by a virus that attacks mainly the upper respiratorytract—the nose, throat and bronchi and rarely also the lungs. Theinfection usually lasts for about a week. It is characterized by suddenonset of high fever, myalgia, headache and severe malaise,non-productive cough, sore throat, and rhinitis. Most people recoverwithin one to two weeks without requiring any medical treatment.However, in the very young, the elderly and people suffering frommedical conditions such as lung diseases, diabetes, cancer, kidney orheart problems, influenza poses a serious risk. In these people, theinfection may lead to severe complications of underlying diseases,pneumonia and death. Annual influenza epidemics are thought to result inbetween three and five million cases of severe illness and between250,000 and 500,000 deaths every year around the world.

Influenza virus is a member of Orthomyxoviridae family. There are threesubtypes of influenza viruses, designated influenza A, influenza B, andinfluenza C. The influenza virion contains a segmented negative-senseRNA genome, which encodes the following proteins: hemagglutinin (HA),neuraminidase (NA), matrix (MI), proton ion-channel protein (M2),nucleoprotein (NP), polymerase basic protein 1 (PBI), polymerase basicprotein 2 (PB2), polymerase acidic protein (PA), and nonstructuralprotein 2 (NS2). The HA, NA, MI, and M2 are membrane associated, whereasNP, PBI, PB2, PA, and NS2 are nucleocapsid associated proteins. The MIprotein is the most abundant protein in influenza particles. The HA andNA proteins are envelope glycoproteins, responsible for virus attachmentand penetration of the viral particles into the cell. Specifically, HAbinds the influenza virus to cells with sialic acid-containing onsurface structures on their membranes.

Both HA and NA proteins are the sources of the major immunodominantepitopes for virus neutralization and protective immunity, making themimportant components for prophylactic influenza vaccines. The geneticmakeup of influenza viruses allows frequent minor genetic changes, knownas antigenic drift. Thus, the amino acid sequence of the major antigensof influenza, particularly HA, is highly variable across groups,subtypes and strains. For this reason, current seasonal influenzavaccines need to be revised every 1-3 years to account for mutations inHA and NA proteins (antigenic drift). A further limitation of thecurrent vaccine approach is that the influenza strains used in thevaccine are selected by the WHO/CDC based on the agencies' best guess asto the prevalent influenza strains for the upcoming flu season. Oftentimes, the guess is not accurate, and the vaccine strains do not matchthe seasonal influenza strains, limiting the effectiveness of theseasonal vaccines. Seasonal vaccines are also not designed to provideprotection against pandemic strains that can result from antigen shift.Further, as the name suggests, seasonal vaccines must be administeredevery year.

Pandemic outbreaks of influenza are caused by the emergence of apathogenic and transmissible virus to which the human population isimmunologically naive. Because the virus is new, the human populationhas little to no immunity against it. The virus spreads quickly fromperson-to-person worldwide. Three times in the last century, theinfluenza A viruses have undergone major genetic changes mainly in theirH-component, resulting in global pandemics and large tolls in terms ofboth disease and deaths. The most infamous pandemic was “Spanish Flu”which affected large parts of the world population and is thought tohave killed at least 40 million people in 1918-1919. More recently, twoother influenza A pandemics occurred in 1957 (“Asian influenza”) and1968 (“Hong Kong influenza”) and caused significant morbidity andmortality globally. In contrast to current influenza epidemics, thesepandemics were associated with severe outcomes also among healthyyounger persons, albeit not on such a dramatic scale as the “Spanishflu” where the death rate was highest among healthy young adults. Morerecently, limited outbreaks of a new influenza subtype A (H1N1) directlytransmitted from swine to humans have occurred in Mexico in 2009 and arebeing detected in an increasing number of countries. Currently, themortality rate associated with swine-origin H1N1 influenza virusesappears to be similar to that of seasonal influenza strains. However,increased surveillance and detection of swine-origin H1N1 influenzacould push the mortality rates higher. Due to antigenic drift, and evenmore dramatic alterations known as antigenic shift, pandemic influenzaantigens (e.g., the HA amino acid sequence of the pandemic strain) arehighly unpredictable. Thus, vaccines have traditionally been unavailableuntil the later stages of a pandemic.

There is an unmet need for influenza vaccines that can better addressthe current problems of antigenic drift, antigenic shift, and virusmismatch by providing broader protection against multiple influenzastrains, including both seasonal and pandemic strains. There is also anunmet need for influenza vaccines that provide longer lasting immunity,particularly vaccines that would not have to be administered every year.

In some embodiments, the present disclosure provides immunogeniccompositions that direct the immune response to highly conserved areas,surface exposed areas of the viral proteins, e.g., the HA and/or NAproteins. In some embodiments, the immunogenic compositions additionallycomprise the M2 ectodomain of the virus. In yet another embodiment, theimmunogenic compositions additionally comprise additional influenzaproteins including internal virus proteins, e.g., the M1, NEP, NS1, NS2,PA, PB1, and PB2 proteins. Specifically, by mutating (e.g.,substituting) hypervariable amino acid residues and/or generatingpolypeptides comprising highly conserved amino acid sequences, thecompositions and methods described herein can be used to induce animmune response against different strains of influenza, including futurestrains that may develop due to antigenic shift.

In one embodiment, the present disclosure provides immunogeniccompositions comprising one or more polypeptides derived from influenzaproteins, wherein at least one hypervariable amino acid residue isreplaced by a conserved, non-hypervariable amino acid residue. In oneembodiment, the non-hypervariable amino acid residue is selected fromamino acid residues with non-polar or neutral side charge. In oneembodiment, the non-hypervariable amino acid residue is selected fromalanine, glycine, valine, leucine, isoleucine and methionine.

In one embodiment, the present disclosure provides immunogeniccompositions comprising one or more polypeptides derived from influenzaproteins, wherein at least one hypervariable amino acid residue isreplaced by an amino acid residue that is a conserved,hypervariable-substitute. In one embodiment, thehypervariable-substitute is selected from amino acid residues withnon-polar or neutral side charge. In one embodiment, thehypervariable-substitute is selected from alanine, glycine, valine,leucine, isoleucine and methionine.

In some embodiments, the immunogenic composition comprises an influenzaprotein or polypeptide having a highly conserved regions as describedherein. In some embodiments, the immunogenic composition comprises aninfluenza protein or polypeptide having a highly conserved regionsannotated in any one of SEQ ID NOs: 171-193. In some embodiments, theprotein or polypeptide comprises a non-hypervariable amino acid residueat an amino acid residue that is a hypervariable amino acid residue asannotated in any one of SEQ ID NOs: 171-193. In some embodiments, theprotein or polypeptide comprises a hypervariable-substitute at an aminoacid residue that is a hypervariable amino acid residue as annotated inany one of SEQ ID NOs: 171-193.

Influenza A

In some embodiments, the methods and compositions described hereintarget influenza A. Influenza A virus is both best characterized and themost serious threat to public health, capable of inducing massiveepidemics or pandemics.

In some embodiments, the methods and compositions described hereincomprise a recombinant viral protein derived from influenza A. In someembodiments, the viral protein of an influenza A virus is selected fromsubtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15, or H16. In some embodiments, the influenza virus is selected fromthe group consisting of H1N1, H3N2, H5N1, and H7N9. In some embodiments,the type A virus is a seasonal strain, such as, /Texas/36/1991,A/Singapore/1986, A/New Caledonia/20/1999, A/Solomon Islands/03/2006,A/Brisbane/59/2007, or A/Wisconsin/67/2005. In some embodiments, thetype A virus is a pandemic strain such as A/California/07/2009,A/California/04/2009, A/Belgium/145/2009, A/South Carolina/01/1918, orA/New Jersey/1976.

Influenza B

In some embodiments, the methods and compositions described hereintarget influenza B. Influenza B viruses generally mutate slower thaninfluenza A viruses.

In some embodiments, the methods and compositions described hereincomprise a recombinant viral protein derived from influenza B. In someembodiments, the viral protein of an influenza B virus is selected froma Yamagata lineage strain or a Victoria lineage strain. In someembodiments, the viral protein of an influenza B virus is selected fromB/Hong Kong/330/2001, B/Hong Kong/05/1972, B/Lee/40,B/Massachusetts/02/2012, B/Panama/45/1990, B/Singapore/222/79,B/Victoria/02/1987, B/Yamagata/16/1988, or B/Brisbane/60/2008.

Hemagglutinin (HA)

In some embodiments, an immunogenic composition described hereincomprises a hemagglutinin (HA) recombinant protein, polypeptide or both.In some embodiments, the HA recombinant protein comprises anon-hypervariable amino acid substituted for a hypervariable amino acidresidue. In some embodiments, the HA recombinant protein comprises anon-hypervariable amino acid replaced with an amino acid that is ahypervariable-substitute. In some embodiments, the HA polypeptidecomprises a highly conserved region of amino acid sequences.

HA is a glycoprotein on the surface of influenza virus responsible forinteraction of the virus with host cell receptors. HA proteins on thevirus surface are trimers of hemagglutinin protein monomers that areenzymatically cleaved to yield amino-terminal HA1 and carboxy-terminalHA2 polypeptides. The globular head consists exclusively of the majorportion of the HA1 polypeptide, whereas the stem that anchors thehemagglutinin protein into the viral lipid envelope is comprised of HA2and part of HAL The globular head of a hemagglutinin protein includestwo domains: the receptor binding domain (RBD), an ^(˜)148-amino acidresidue domain that includes the sialic acid-binding site, and thevestigial esterase domain, a smaller ^(˜)75-amino acid residue regionjust below the RBD. The top part of the RBD adjacent to the 2,6-sialicacid recognition sites includes a large region (amino acids 131-143,170-182, 205-215 and 257-262, 1918 numbering) (referred to herein as theRBD-A region) of over 6000 A2 per trimer that is 95% conserved betweenA/South Carolina/1/1918 (1918 SC) and A/California/04/2009 (2009 CA)pandemic strains. The globular head includes several antigenic sitesthat include immunodominant epitopes. Examples include the Sa, Sb, Ca1,Ca2 and Cb antigenic sites (see, for example, Caton A J et al, 1982,Cell 31, 417-427). The RBD-A region includes the Sa antigenic site andpart of the Sb antigenic site.

H1N1

In some embodiments, the immunogenic composition comprises an HArecombinant protein or polypeptide derived from H1N1. In someembodiments, the recombinant H1N1 HA protein or polypeptide comprises anon-hypervariable amino acid residue at an amino acid position selectedfrom Table 1, or any combination thereof. In some embodiments, therecombinant H1N1 HA protein or polypeptide comprises an amino acid thatis a hypervariable-substitute at an amino acid position selected fromTable 1, or any combination thereof.

TABLE 1 List of Hypervariable Amino Acid Residues in H1N1 HA Protein 13T  14T  52D  53K  78I  86S  88S 101N 114N 137T 145S 147K 155H 158A159K 160S 163K 178L 179N 180Q 185D 187G 195G 200S 202T 203A 214A 220T222R 225K 228K 233T 239D 241E 251V 256K 273T 274M 275E 277N 278A 287T288P 293N 300E 315I 319K 331L 338V 362V 382L 391K 415N 419K 468N 490N491T 516K 544V *residue numbering based on straight numbering of SEQ IDNO: 1. SEQ ID NO: 1 indicates these residues in bold.

In some embodiments, the recombinant H1N1 HA polypeptide comprises ahighly conserved region of amino acid sequences. In some embodiments,the highly conserved region of amino acid sequences is selected fromTable 2, or any combination thereof.

TABLE 2 Highly Conserved Regions in H1N1 HA Protein  GYHANNST NVTVTHSSWSYIVE QSRGLFGAIAGF (SEQ ID NO 7) (SEQ ID NO 9) (SEQ ID NO 11)(SEQ ID NO 13) QGSGYAAD ITNKVNS WTYNAELL GCFEFYH (SEQ ID NO 15)(SEQ ID NO 17) (SEQ ID NO 19) (SEQ ID NO 21) LGNPEC EGGWTG LLENER(SEQ ID NO 23) (SEQ ID NO 25) (SEQ ID NO 27)H3N2

In some embodiments, the immunogenic composition comprises an HArecombinant protein or polypeptide derived from H3N2. In someembodiments, the recombinant H3N2 HA protein or polypeptide comprises anon-hypervariable amino acid residue at an amino acid position selectedfrom Table 3, or any combination thereof. In some embodiments, therecombinant H3N2 HA protein or polypeptide comprises an amino acid thatis a hypervariable-substitute at an amino acid position selected fromTable 3, or any combination thereof.

TABLE 3 List of Hypervariable Amino Acid Residues in H3N2 HA Protein  7L 26T  73Q 110Y 151T 172H 189Q 214S 242I 296E 391D 494I  9Y  41I  78E117D 153S 173L 202G 215S 243P 315R 394N 495G  14V  47N  91Q 137N 154A174N 205K 218I 245R 327Q 400L 500G  16A  49R  94G 138N 156I 175F 206D219T 277R 328S 402G 505N  18K  61N  98K 140S 158R 176K 208I 228A 278S342K 422I 506V  19L  64I  99K 144T 160N 179A 209F 238R 291G 362M 466K509D  22Y  66E 107S 147T 161S 187N 212A 239I 292K 363V 468K 522E  25S 69D 108K 149N 171T 188E 213Q 241N 294K 377R 469K 545V 546A 560I 561R562C 563N 559N *residue numbering based on straight numbering of SEQ IDNO: 3. SEQ ID NO: 3 indicates these residues in hold

In some embodiments, the recombinant H3N2 HA polypeptide comprises ahighly conserved region of amino acid sequences. In some embodiments,the highly conserved region of amino acid sequences is selected fromTable 4, or any combination thereof.

TABLE 4 Highly Conserved Regions in H3N2 HA Protein LCLGHHA GNLIAPRGYFLKLATGMRN FGAIAGF (SEQ ID NO 61) (SEQ ID NO 63) (SEQ ID NO 65) IENGWEG(SEQ ID NO 67) KFHQIEKEF DLTDSEM LRENAED (SEQ ID NO 69) (SEQ ID NO 71)(SEQ ID NO 73)Influenza B

In some embodiments, the immunogenic composition comprises an HArecombinant protein or polypeptide derived from influenza B. In someembodiments, the recombinant influenza B HA protein or polypeptidecomprises a non-hypervariable amino acid residue at an amino acidposition selected from Table 5, or any combination thereof. In someembodiments, the recombinant influenza B HA protein or polypeptidecomprises an amino acid that is a hypervariable-substitute at an aminoacid position selected from Table 5, or any combination thereof.

TABLE 5 List of Hypervariable Amino Acid Residues in Influenza B HAProtein  55H  63E  71K  73L  86K  88T  90K  91I  95R  96V 123P 131H 132I137H 141N 144N 151K 152I 161I 163N 164G 165N 177K 178N 180K 181N 183T187P 188L 190I 195I 197T 213E 217A 218K 224K 245G 248N 267V 270S 277T282I 314K 328E 494E 513R 520D 566I 570V *residue numbering based onstraight numbering of SEQ ID NO: 5. SEQ ID NO: 5 indicates theseresidues in bold.

In some embodiments, the recombinant influenza B HA polypeptidecomprises a highly conserved region of amino acid sequences. In someembodiments, the highly conserved region of amino acid sequences isselected from Table 6, or any combination thereof.

TABLE 6 Highly Conserved Regions in Influenza B HA Protein VKTATQGNCTDLDVAL TSGCFPIMH NLLRGYE EVNVTG (SEQ ID NO 95) DRTKIRQL(SEQ ID NO 99) (SEQ ID NO 194) (SEQ ID NO 97) TMAWAVP EDGGLPQS LPLIGEADYGGLNKSKP (SEQ ID NO 101) GRIWDYM CLHE YYTG (SEQ ID NO 103)(SEQ ID NO 105) (SEQ ID NO 107) CPIWVKTPL GFFGAIAGF AGWHGYTSHGAHGAVAADLKSTQEA (SEQ ID NO 109) LEGGWEGM (SEQ ID NO 113 (SEQ ID NO 115)(SEQ ID NO 111) KITKNLNSLSELE KNLQRLS EILELDEK IGNGCFETKH(SEQ ID NO 117) (SEQ ID NO 119) VDDLRADT KCNQTCLD ISSQIELA(SEQ ID NO 123) VLLSNEGI INSEDEHL LALERKLK KMLGPSA (SEQ ID NO 121)AGEFSLPTFD HTILLYYSTA SLNITAASL ASSLAVTLM (SEQ ID NO 125)(SEQ ID NO 127)Neuraminidase (NA)

In some embodiments, an immunogenic composition described hereincomprises a neuraminidase (NA) recombinant protein, polypeptide or both.In some embodiments, the NA recombinant protein comprises anon-hypervariable amino acid substituted for a hypervariable amino acidresidue. In some embodiments, the NA recombinant protein comprises anon-hypervariable amino acid replaced with an amino acid that is ahypervariable-substitute. In some embodiments, the NA polypeptidecomprises a highly conserved region of amino acid sequences.

NA is an enzyme found on the surface of influenza that enables the virusto be released from the host cells. Neuraminidases are enzymes thatcleave sialic acid groups from glycoproteins and are required for virusreplication. The NA protein also functions during entry of virus intothe respiratory tract. The epithelial cells are bathed in mucus, acomplex protective coating that contains may sialic acid-containingglycoproteins. When influenza virions enter the respirator tract, theyare trapped in mucus where they bind sialic acids. This interactionwould prevent the viruses from binding to a susceptible cell were it notfor the action of the NA protein. When a virus particle encounters acell, it binds the sialic acid-containing receptor and is rapidly takeninto the cell before the NA protein can cleave the carbohydrate from thecell surface.

The NA is a tetramer of four identical polypeptides. Each polypeptidecontains about 470 amino acids arranged in four domains, an N-terminalcytoplasmic sequence, followed by a membrane-anchoring hydrophobictransmembrane domain and a thin stalk of variable length, ending in aglobular “head” domain that carries the enzyme active site. Crystalstructures of NA encompass the catalytically active heads, eitherproteolytically cleaved from the virus or engineered as a solublesecreted protein. The intact NA has not been crystallized, but acryoelectron microscopy study of the X-31 (A/Aichi/68, H3N2) reassortantvirus has revealed considerable detail at near atomic resolution. Thestructure confirms that the N2 NA protrudes slightly further than the HAfrom the viral membrane, that there are 40-50 NA spikes per virion, andthat these occur in clusters amid 300-400 HA spikes on an average sizedvirion of diameter 120 nm.

H1N1

In some embodiments, the immunogenic composition comprises an NArecombinant protein or polypeptide derived from H1N1. In someembodiments, the recombinant H1N1 NA protein or polypeptide comprises anon-hypervariable amino acid residue at an amino acid position selectedfrom Table 7, or any combination thereof. In some embodiments, therecombinant H1N1 NA protein or polypeptide comprises an amino acid thatis a hypervariable-substitute at an amino acid position selected fromTable 7, or any combination thereof.

TABLE 7 List of Hypervariable Amino Acid Residues in H1N1 NA Protein 13I  14C  15M  16T  19M  20A  21N  23I  34I  40L  42N  44N  45Q  46I 47E  48T  52S  53V  59N  64Q  70S  74F  75A  77G  78Q  79S  80V  81V 82S  84K  86A  93P  94V 101S 106I 126P 130R 149I 157T 163I 166V 173R188I 189N 200N 214D 220R 221N 222N 232A 234V 241I 248D 249G 250Q 257R263I 264V 267V 269M 270N 274Y 275H 285S 286S 287E 288I 289T 311E 314I321V 329N 331K 332T 336G 339S 340S 341N 344N 351F 354G 365I 366S 367S369K 382G 385N 386N 389I 393I 395G 397N 398E 416D 427I 430R 432E 449N450S 452T 453V 454G *residue numbering based on straight numbering ofSEQ ID NO: 2. SEQ ID NO: 2 indicates these residues in bold.

In some embodiments, the recombinant H1N1 NA polypeptide comprises ahighly conserved region of amino acid sequences. In some embodiments,the highly conserved region of amino acid sequences is selected fromTable 8, or any combination thereof.

TABLE 8 Highly Conserved Regions in H1N1 NA Protein  MNPNQKIITIGSRIGSKGDVFV REPFISCS TFFLTQGAL (SEQ ID NO 29) (SEQ ID NO 31)(SEQ ID NO 33) LNDKHSNGT (SEQ ID NO 35) KDRSPYR FESVAWSASACHDGWLTIGISGPD GAVAVLKY (SEQ ID NO 37) (SEQ ID NO 39) (SEQ ID NO 41)(SEQ ID NO 155) ILRTQESEC YEECSCYPD CVCRDNWHGS NGVWIGRTKS (SEQ ID NO 43)(SEQ ID NO 45) NRPWVSFNQNL (SEQ ID NO 49) (SEQ ID NO 47) GFEMIWDPNGWTWSGYSGSFV RPCFWVEL WTSGSSISFCGV (SEQ ID NO 51) QHPELTGL (SEQ ID NO 55)(SEQ ID NO 57) (SEQ ID NO 53) WSWPDGAELPF (SEQ ID NO 59)H3N2

In some embodiments, the immunogenic composition comprises an NArecombinant protein or polypeptide derived from H3N2. In someembodiments, the recombinant H3N2 NA protein or polypeptide comprises anon-hypervariable amino acid residue at an amino acid position selectedfrom Table 9, or any combination thereof. In some embodiments, therecombinant H3N2 NA protein or polypeptide comprises an amino acid thatis a hypervariable-substitute at an amino acid position selected fromTable 9, or any combination thereof.

TABLE 9 List of Hypervariable Amino Acid Residues in H3N2 NA Protein 16T  18S  23F  26I  30I  40Y  41E  42F  43N  44S  45P  46P  51M  52L 56T  62I  73I  75K  81L  82A  93N 126P 127D 140L 143V 147D 149V 150R155Y 161N 172K 176I 194V 197D 199K 208N 215I 216V 220K 221E 249K 263V265T 267T 303V 307I 310Y 313V 329N 331S 332S 336H 338L 339D 344E 346G356D 367S 369K 370L 372S 380I 381E 385N 386P 387N 392I 399D 400R 401G402N 416S 432E 435E 437L 464I 468P *residue numbering based on straightnumbering of SEQ ID NO: 4. SEQ ID NO: 4 indicates these residues inbold.

In some embodiments, the recombinant H3N2 NA polypeptide comprises ahighly conserved region of amino acid sequences. In some embodiments,the highly conserved region of amino acid sequences is selected fromTable 10, or any combination thereof.

TABLE 10 Highly Conserved Regions in H3N2 NA Protein  QFALGQGTT AWSSSSCLRTQESEC (SEQ ID NO 75) (SEQ ID NO 77) (SEQ ID NO 79) EECSCYP CSGLVGDTPRGVKGWAFD (SEQ ID NO 81) (SEQ ID NO 83) (SEQ ID NO 85) NRCFYVELIRGVFCGTSGTYG GSWPDGA (SEQ ID NO 87) (SEQ ID NO 89) (SEQ ID NO 91)Influenza B

In some embodiments, the immunogenic composition comprises an NArecombinant protein or polypeptide derived from influenza B. In someembodiments, the recombinant influenza B NA protein or polypeptidecomprises a non-hypervariable amino acid residue at an amino acidposition selected from Table 11, or any combination thereof. In someembodiments, the recombinant influenza B NA protein or polypeptidecomprises an amino acid that is a hypervariable-substitute at an aminoacid position selected from Table 11, or any combination thereof.

TABLE 11 List of Hypervariable Amino Acid Residues in Influenza B NAProtein 42P 45I 49T 61Q 65R 67A 68T 73L 74L 107T 121V 126N 149G 172I187K 199N 205V 220N 221N 236N 245S 249V 296R 321D 330N 341D 343D 344K359K 372K 374E 385G 390A 393D 396A 397F 402V 403S 405K 437E 464D 466A*residue numbering based on straight numbering of SEQ ID NO: 6. SEQ IDNO: 6 indicates these residues in bold.

In some embodiments, the recombinant influenza B NA polypeptidecomprises a highly conserved region of amino acid sequences. In someembodiments, the highly conserved region of amino acid sequences isselected from Table 12, or any combination thereof.

TABLE 12 Highly Conserved Regions in Influenza B NA Protein HFALTHYAAQPGDRNKLRHL AWSGSACHDG KYGEAYT (SEQ ID NO 131) (SEQ ID NO 133)(SEQ ID NO 135) DTYHSY (SEQ ID NO 137) LRTQESACNCI CRFLKIREGR HTEECTCGFAYTAKRPFVKL (SEQ ID NO 139) (SEQ ID NO 141) (SEQ ID NO 143)(SEQ ID NO 145) KGGFVHQR GRWYSRT EPGWYSFGFE EMVHDGG (SEQ ID NO 147)(SEQ ID NO 149) (SEQ ID NO 151) (SEQ ID NO 153) ALLISPHRFGE(SEQ ID NO 129)M2 Ectodomain

In some embodiments, an immunogenic composition described hereincomprises an M2 ectodomain (M2e) recombinant protein, polypeptide orboth. In some embodiments, the M2e recombinant protein comprises anon-hypervariable amino acid substituted for a hypervariable amino acidresidue. In some embodiments, the M2e recombinant protein comprises anon-hypervariable amino acid replaced with an amino acid residue that isa hypervariable-substitute. In some embodiments, the M2e polypeptidecomprises a highly conserved region of amino acid sequences.

The M2 protein is a surface protein on the influenza virion encoded bythe M segment. The M segment encodes M1 from unspliced mRNA and M2protein by mRNA splicing. M2 forms homotetramers and possesses ionchannel activity that allows for acidification of the inside of thevirion during endocytosis and facilitates the dissociation of the matrixprotein M1 from viral ribonucleoprotein complexes. The M2e, which is theexposed portion of the M2 protein found on the virion membrane, ishighly conserved among influenza strains. Accordingly, the M2e proteinis a target for universal influenza vaccine approaches.

In some embodiments, M2e protein sequences are obtained and alignedusing a method described herein (e.g., the Dawn method) to identifyhypervariable amino acid residues subject to antigenic shift/drift, andhighly conserved regions of amino acid sequences.

In some embodiments, the immunogenic composition comprises an M2erecombinant protein or polypeptide derived from H1N1. In someembodiments, the recombinant H1N1 M2e protein or polypeptide comprises anon-hypervariable amino acid residue at a hypervariable amino acidresidue, or any combination thereof. In some embodiments, therecombinant H1N1 M2e polypeptide comprises a highly conserved region ofamino acid sequences, or any combination thereof.

In some embodiments, the immunogenic composition comprises an M2erecombinant protein or polypeptide derived from H3N2. In someembodiments, the recombinant H3N2 M2e protein or polypeptide comprises anon-hypervariable amino acid residue at a hypervariable amino acidresidue, or any combination thereof. In some embodiments, therecombinant H3N2 M2e polypeptide comprises a highly conserved region ofamino acid sequences, or any combination thereof.

In some embodiments, the immunogenic composition comprises an M2erecombinant protein or polypeptide derived from influenza B. In someembodiments, the recombinant influenza B M2e protein or polypeptidecomprises a non-hypervariable amino acid residue at a hypervariableamino acid residue, or any combination thereof. In some embodiments, therecombinant influenza B M2e polypeptide comprises a highly conservedregion of amino acid sequences, or any combination thereof.

Additional Influenza Proteins

In some embodiments, an immunogenic composition described hereincomprises at least one additional influenza protein, polypeptides orboth. In some embodiments the at least one additional influenza proteinis selected from NP, M1, PA, PB2, PB2, NS1, and NS2. In someembodiments, the additional influenza protein comprises anon-hypervariable amino acid substituted for a hypervariable amino acidresidue. In some embodiments, the additional influenza protein comprisesa non-hypervariable amino acid replaced with an amino acid residue thatis a hypervariable-substitute. In some embodiments, the additionalinfluenza protein comprises a highly conserved region of amino acidsequences.

The nucleoprotein molecules encapsidate the viral single-stranded RNAs.Nucleoprotein molecules also participate in the nuclear import andexport of vRNPs and viral replication, and interact with host proteins.The influenza viral polymerase (P complex) is a heterotrimer of subunitsPA, PB1 and PB2. The P complex carries out mRNA transcription andreplication of the influenza virus. The PA subunit N domain has acation-dependent endonuclease active-site core; the catalytic residuesHis41, Glu80, Asp108 and Glu119 are conserved among influenza A subtypesand strains. The nonstructural protein NS1 binds double-stranded RNA(dsRNA) in a non-sequence specific manner. The NS1 protein has aconserved residue, Arg39 that interact with dsRNA. Accordingly, theadditional influenza proteins are also targets for universal influenzavaccine approaches.

In some embodiments, the additional influenza protein sequences areobtained and aligned using a method described herein (e.g., the Dawnmethod) to identify hypervariable amino acid residues subject toantigenic shift/drift, and highly conserved regions of amino acidsequences.

In some embodiments, the immunogenic composition comprises an additionalinfluenza protein or polypeptide derived from H1N1. In some embodiments,the recombinant H1N1 protein or polypeptide comprises anon-hypervariable amino acid residue at a hypervariable amino acidresidue, or any combination thereof. In some embodiments, therecombinant H1N1 polypeptide comprises a highly conserved region ofamino acid sequences, or any combination thereof.

In some embodiments, the immunogenic composition comprises an additionalinfluenza protein or polypeptide derived from H3N2. In some embodiments,the recombinant H3N2 protein or polypeptide comprises anon-hypervariable amino acid residue at a hypervariable amino acidresidue, or any combination thereof. In some embodiments, therecombinant H3N2 polypeptide comprises a highly conserved region ofamino acid sequences, or any combination thereof.

In some embodiments, the immunogenic composition comprises an additionalinfluenza protein or polypeptide derived from influenza B. In someembodiments, the recombinant influenza B protein or polypeptidecomprises a non-hypervariable amino acid residue at a hypervariableamino acid residue, or any combination thereof. In some embodiments, therecombinant influenza B polypeptide comprises a highly conserved regionof amino acid sequences, or any combination thereof.

In some embodiments, the immunogenic composition comprises an additionalinfluenza protein or polypeptide having a highly conserved regions asannotated in any one of SEQ ID NOs: 171-193. In some embodiments, theprotein or polypeptide comprises a non-hypervariable amino acid residueat an amino acid residue that is a hypervariable amino acid residue asannotated in any one of SEQ ID NOs: 171-193. In some embodiments, theprotein or polypeptide comprises a hypervariable-substitute at an aminoacid residue that is a hypervariable amino acid residue as annotated inany one of SEQ ID NOs: 171-193.

Methods for Identifying Hypervariable and Conserved Influenza Residues

In some embodiments, the present disclosure provides methods foridentifying hypervariable and conserved residues in an influenza viralprotein between types and/or subtypes of strains. In some embodiments,the hypervariable amino acid residues identified by the methodsdescribed herein are substituted with a non-hypervariable amino acid(e.g., alanine). In some embodiments, the hypervariable amino acidresidues identified by the methods described herein are substituted withan amino acid residue that is a hypervariable-substitute (e.g.,alanine). In some embodiments, the highly conserved regions of aminoacids are used to generate polypeptides for peptide vaccines and/or astargets of nucleic acids.

In some embodiments, the present disclosure provides methods forevaluating the role of hypervariable and conserved residues on theability to induce an immune response (e.g., production of antibodies).

Sequence Alignment—Dawn Method

Protein sequence evolutionary conservation analysis improvesunderstanding of protein structure, function, and disease. Multiplesequence alignments of different isolates, orthologs, paralogs, andfunctional domains provide essential insights into protein function andstructure. Evolutionary conservation level is directly correlated withlikelihood of missense mutations' functional impact.

Missense mutations are typically either deleterious or neutral inregards to function impact. Deleterious mutations experience negativeselection. Neutral mutations are not positively or negatively selectedand can drift through populations. A few mutations experience positiveselection and become fixed within populations. Aligning sequences fromdifferent species enable the estimation of residue functional importancebased on sequence divergence of evolutionarily related proteins. Alignedresidues that are identical are composed of a combination offunctionally important residues and residues not observed to change dueto stochastic chance. Aligned residue positions that are different canrepresent (1) functionally neutral residues, (2) positions that allowlimited conservative residue changes of similar amino acid residues, and(3) positions with alignment errors (this varies by alignment toolused).

A protein enzyme typically consists of a globular domain with aconserved inner core with non-conserved residues observed on the solventexposed surface. Protein folding includes structures like random coils,alpha helices, beta sheets, and loops/turns. Unlike a ball of yarn,protein peptide strands fold into the tertiary structure with thepeptide strand traversing into the interior until typically turning in asolvent exposed loop. Residues in the inner core are typically conservedwith amino acid substitutions likely impacting protein folding,structure, and/or function. These interior segments are typically whatmotif signature models such as, Profile analysis, Psi-Blast, and HAMMR,are derived from or trained on. See e.g., Gribskov, M. et al., Proc.Natl. Acad. Sci., 84: 4355-4358 (1987); Altschul, S. F., et al., Nucl.Acids Res. 25:3389-3402 (1997); Eddy, S. R., Bioinformatics, 14:755-763(1998). Ideally, these are the segments that should be aligned in amultiple sequence alignment without gapping allowed within each segment.Small insertions and gaps are observed in exterior turns/loops.Pascarella, S., and P. Argos, P., J Mol. Biol., 224: 461-471 (1992).

Multiple sequence alignment of protein sequences provides numerousinsights into protein structures and functions. Available solutions forgenerating multiple sequence alignments is slow, and the resultingalignments are plagued by frequent over gapping. Scientists routinelyrealign sub-segments within alignments to enhance alignment quality.Algorithm developers have treated protein sequences as text strings forcomparisons. Some advanced algorithms include knowledge extracted frommotifs, profiles, and structures.

In some embodiments, protein sequences are aligned using the Dawnmethod.

The Dawn multiple sequence alignment and conservation analysis tool usesconserved residues as anchors such that evolutionarily related sequencescan be added to the alignment incrementally. This approach reduces thecomplexity of creating multiple sequence alignment, or of comparingevery sequence to every other sequence. This works for bothevolutionarily close or distant sequences and combinations of both.

The ability to identify distant orthologs is directly correlated withthe proportion of essential residues in a protein. Doolittle, R. F., OFURFS AND ORFS: A Primer on How to Analyze Derived Amino Acid Sequences:University Science Books, (1986), characterized sequence alignmentsbelow 25% identity as being the “Twilight Zone”—a limit on sequencealignment approaches. Below 20% identify is termed the “Midnight Zone”,an accepted theoretical limit to sequence analysis techniques.

Dawn aligns sequences based on the Divergence Model of proteinevolution, and can align and characterize large numbers of relatedprotein sequences rapidly. Using this tool, a performance improvement ofat least two orders of magnitude improvement over current methods. Dawnis applied to three pressing challenges: identification of antiviraltargets for therapeutics, multigene family alignment, and analysis ofhuman missense mutations (variants). Dawn implements two concepts of (1)conserved core segments and (2) insertions in loops. Using the sequenceanalysis technique, Dawn is able to align some paralogs deep into thesequence alignment Twilight and Midnight Zones.

In some embodiments, the workflow for multiple sequence alignmentstrategy comprises:

-   1. Identify highly conserved protein segments and use these as    vertical segments throughout the multiple sequence alignment;-   2. Place insertions and gaps in candidate loop segments to align    conserved segments. To minimize alignment gaps, align insertions and    gaps in a loop region in overlapping alignment positions unless    local sequence identity indicate two likely independent mutation    events have likely occurred;-   3. Residues in two homologs share high sequence identity between    conserved segments that are ordered within a domain. Unrelated    sequences can share common simple sequence motifs, but these can be    ignored;-   4. Conserved segments can be approximated by common k-mers between    sequences. Multiple homologs will share a common set of ordered    k-mers. Multiple unrelated sequences will not share ordered k-mer    sequences outside of expected random sharing.

In some embodiments, the following definitions are used to defineconserved segments:

-   -   Ai=Multiple sequence alignment position, i, for sequence of        interest;    -   C(Ai)=−V with V>0—nonconserved position with V different amino        acids observed at this alignment position;    -   C(Ai)=0—nonconserved position with residue observed missing in        sequences for this gene;    -   C(Ai)=1—conserved positions for all sequences for this gene for        organisms of the same taxonomic class;    -   C(Ai)=1.T—conserved position for all sequences for this gene for        taxonomic class of this sequence and T-1 additional taxonomic        classes; and    -   C(Ai)=V with V>1—conserved position with residue conserved in        all sequences for V genes.

In some embodiments, the following definitions are used to defineconserved variable or non-conserved segments:

-   -   V(Ai)=number of nonconserved residues observed at alignment        position, i, for the taxonomic class of interest. Allowable        conservative substitutions defined by Bottema were used to        define observed nonconservative substitutions. Bottema, C. D.        K., et al., Am J Hum Genet, (49):820-838 (1991).

In some embodiments, an algorithm, MSAQ-compute.py (Multiple sequencealignment quality compute), developed in Python, is used to evaluate thequality of multiple sequence alignments. The algorithm accepts an MSA inClustal format as an input, as well as optional parameters for thenumber of residues that should not be scored at the beginning and end ofthe alignment. This accommodates cases of partial sequence overlap andavoids imposing a penalty for otherwise good alignments with excessresidues at the beginning or end. The algorithm generates an index ofall scored positions within the MSA input file and tallies reportedresidues at each position to generate a consensus sequence for thealignment.

For each sequence in the MSA, the algorithm computes the number ofresidues that match the consensus sequence, the number of residues thatare different from the corresponding position in the consensus sequence(mismatch), the total number of gap characters in the aligned sequence,and the total number of unique gaps in the aligned sequence. Thesevalues are reported in a “details” file generated by the algorithm.Additionally, these values are averaged across all sequences in the MSAto generate average match and average mismatch metrics.

The average length of all gaps in the MSA is also reported as well asthe total number of gaps present in the alignment (summed across allsequences). Finally, based on the rationale that any gaps in a highquality alignment should overlap (i.e. input sequences should havealignment gaps at roughly the same positions), the number ofnon-overlapping gaps is computed. To generate this value, all gaps inthe alignment are mapped to positions in the consensus sequence togenerate ranges of gap positions. The number of such non-overlappingranges is reported.

In some embodiments, viral protein sequences are selected from GenBankfor influenza. For each selected virus protein, subsets are evaluated tomeasure execution runtimes using a single Linux core (noparallelization).

Alanine Scanning

The methods described supra are used to identify hypervariable aminoacid residues. In some embodiments, the importance of a hypervariableamino acid residue for inducing an immune response is determined byalanine scanning.

As described herein, alanine scanning is a technique used to determinethe contribution of a specific wild-type residue to the stability orfunction(s) (e.g., inducing an immune response) of given protein orpolypeptide. The technique involves the substitution of an alanineresidue for a wild-type residue in a polypeptide, followed by anassessment of the stability or function(s) (e.g., inducing an immuneresponse) of the alanine-substituted derivative or mutant polypeptideand comparison to the wild-type polypeptide. In some embodiments, theresidues identified as not critical are further evaluated to modulatethe induction of an immune response. A non-limiting example of suchanalysis is deep mutational scanning. This method allows for theevaluation of large numbers of mutations. Other methods for analyzingthe effect of amino acid residue mutations are known in the art. Forexample, arginine/glutamic acid scanning is employed to study theeffects of bulky, charged amino acid residues on antigen binding. In anembodiment, an arginine amino acid in the hypervariable region isreplaced by glutamic acid.

Inducing T Cell Responses with Highly Conserved Regions

T cell immune response plays an important role in eliciting andmaintaining protective immunity against influenza virus. In a recenthuman study, repeated influenza virus boosted multifunctional memoryCD4+ T cell populations. Specifically, IFN-γ and TNF-α secreting CD4cell population have been shown to boost anti-virus antibody titersafter repeated vaccination, and is correlated with maintenance ofprotective antibody titers. Trieu, M. C., et al., npj Vaccines, 3:37(2018). doi:10.1038/s41541-018-0069-1. Similarly, administration of acombination vaccine comprising trivalent influenza vaccine and a VLPbased vaccine showed enhanced CD8+ and CD4+ immune response, and CD4+T-cell response is correlated with neutralization antibody titers.Skibinski, D. A. G., et al., Sci Rep. 8:18007 (2018).

In some embodiments, the present disclosure provides a polypeptidecomprising highly conserved regions of amino acid sequences within aviral protein. In some embodiments, the conserved region is a continuousstretch of at least 7, 8, 9, 10, 11, or 12 invariant or minimallyvariable amino acid residues. In some embodiments, the polypeptide has100% identity to a highly conserved region provided herein. In someembodiments, the polypeptide has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91% or 90% identity to a highly conserved region provided herein.

In some embodiments, a polypeptide comprising a highly conserved regionis operably linked to at least one additional polypeptide comprising adifferent highly conserved region. In some embodiments, a polypeptidecomprising a highly conserved region is operably linked to at least oneadditional polypeptide comprising the same highly conserved region. Insome embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 or 20 polypeptides comprising highly conserved regions areoperably linked to each other, wherein each polypeptide is the same ordifferent. In some embodiments, at least 10, at least 20, at least 30,at least 40 or at least 50 polypeptides comprising highly conservedregions are operably linked to each other, wherein each polypeptide isthe same or different.

In some embodiments, a polypeptide or polypeptides operably linked toeach other, induce a T cell response, such as virus-specific CD8+ orCD4+ T cell responses. In some embodiments, an virus-specific CD8+ Tcell response comprises CD8+ T cell proliferation or CD8+ T cellcytokine production or both, are induced. In some embodiments, CD8⁺ Tcell cytokine production increases by at least 5% or at least 10% or atleast 15% or at least 20% or at least 25% or at least 30% or at least35% or at least 40% or at least 45% or at least 50%. In someembodiments, the percentage of CD8⁺ T cells among the total T cellpopulation increases by at least 5% or at least 10% or at least 15% orat least 20% or at least 25% or at least 30% or at least 35% or at least40% or at least 45% or at least 50%.

In one embodiment, the disclosure provides a method for eliciting T cellresponse to conserved polypeptides of influenza viruses, the methodcomprising administering to a subject in need thereof an immunogeniccomposition comprising at least one influenza virus polypeptidecomprising high conserved amino acid sequence, wherein the T cellresponse immune response to the highly conserved amino acid sequence iselicited in the subject. In one embodiment, eliciting T cell immuneresponse in a subject comprises stimulating cytokine production (e.g.,IFN-γ or TNF-α).

In another embodiment, eliciting an immune response in a subjectcomprises stimulating virus polypeptide-specific CD4+ or CD8+ T cellactivity, e.g., priming, proliferation and/or survival (e.g., increasingthe effector/memory T cell population). In one aspect, eliciting aT-cell immune response in a subject comprises stimulating virus-specificCD4+ T cell activity (e.g., increasing helper T cell activity). In otheraspects, the CD4+ T cell immune response stimulates cell responses(e.g., increasing antibody production). In some embodiments, enhancing Tcell immune response in a subject comprises stimulating cytokineproduction, stimulating antigen-specific CD8+ T cell responses,stimulating antigen-specific CD4+ helper cell responses, increasing theeffector memory CD62Llo T cell population, stimulating B cell activityor stimulating virus-specific antibody production, or any combination ofthe foregoing responses.

In some embodiments, the enhanced immune response comprises anvirus-specific CD8+ T cell response, wherein the CD8+ T cell responsecomprises an increase in the percentage of effector memory CD62Llo Tcells among CD8+ T cells.

Inducing B Cell Responses by Targeting Hypervariable Amino Acid Residues

Most neutralizing antibodies bind to the loops that surround the virusreceptor binding site and interfere with receptor binding andattachment. Since these loops are highly variable, most antibodiestargeting these regions are strain-specific, and elicit limited,strain-specific immunity. Fully human monoclonal antibodies againstinfluenza virus hemagglutinin with broad cross-neutralizing potency havebeen generated. Functional and structural analysis have revealed thatthese antibodies interfere with the membrane fusion process and aredirected against highly conserved epitopes in the stem domain of theinfluenza HA protein (Throsby et al., Plos One 12(3): 1-15 (2008);Ekiert et al., Science 324:246-251 (2009), US2009/0311265,US2012/0039898, US2014/0120113).

In some embodiments, the present disclosure provides an influenza viralprotein or fragment thereof, wherein hypervariable amino acid residuesare replaced with a non-hypervariable amino acid. Non-hypervariableamino acid residues include, but are not limited to, alanine andglycine. In some embodiments, a non-hypervariable amino acid residue isreferred to as a hypervariable-substitute. In some embodiments, thehypervariable amino acid residues are replaced with alanine, glycine,valine, leucine, isoleucine, and methionine. In some embodiments, thehypervariable amino acid residues are replaced with alanine and glycine.In some embodiments, the hypervariable amino acids are replaced with theexemplary and/or preferred amino acids to preserve the conformation ofthe viral protein and to minimize disruption to adjacent or overlappingconserved regions. In some embodiments, bulky and charged arginine aminoacid residues are replaced with glutamic acid residues. In someembodiments, the polypeptide comprises a fragment of the amino acidsequence of the viral proteins. In some embodiments, the fragmentcomprises the entire amino acid sequence of the viral protein. In someembodiments, viral proteins and fragments thereof can be used incombination.

In some embodiments, the present disclosure provides immunogeniccomposition comprising at least viral protein or fragment thereofwherein 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45,46-50, 51-55, 56-60 hypervariable amino acid residues are replaced withnon-hypervariable amino acid residues. In some embodiments at one, two,three, four, five or more hypervariable amino acids are replaced withnon-hypervariable amino acid residues.

In some embodiments, the present disclosure provides immunogeniccomposition comprising at least viral protein or fragment thereofwherein 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45,46-50, 51-55, 56-60 hypervariable amino acid residues are replaced withamino acid residues that are hypervariable-substitute. In someembodiments at one, two, three, four, five or more hypervariable aminoacids are replaced with an amino acid residue that is ahypervariable-substitute.

In some embodiments, substituting the hypervariable amino acid residueswith non-hypervariable amino acid residues directs the immune responseaway from the residues subject to antigenic drift/shift and induces animmune response to the highly conserved regions of amino acid sequences.By targeting the highly conserved regions, such polypeptides can be usedfor protection against current and yet to exist influenza strains.

In some embodiments, the polypeptides described herein induce a B cellresponse (e.g., antibody production). In some embodiments, the B cellresponse is an antigen-specific antibody response. In some embodiments,the B cell response elicit neutralizing antibodies directed to thehighly conserved regions in the viral protein. In some embodiments, theneutralizing antibodies are neutralizing against multiple strains ofinfluenza viruses.

In another aspect, the disclosure provides a method of directing thespecificity of an B cell immune response in a subject by administeringto a subject an immunogenic composition comprising the viral protein,wherein one or more hypervariable amino acid residues of the virusprotein are replaced with non-hypervariable amino acid residues.

In another embodiment, administration of immunogenic composition havingthe amino acid residue substitution results in the immune response to bedirected to an highly conserved B cell epitope, and thus eliciting oneor more protective neutralizing antibodies. In some embodiments, theneutralizing antibodies provide protective immunity against multiplestrains of influenza virus.

Targeting Highly Conserved Regions with Nucleic Acid Molecules

In some embodiments, the present disclosure provides nucleic acidmolecules having substantial complementarity to a highly conservedregion of amino acid residues. Such nucleic acid molecules are capableof disrupting the transcription and/or translation of a viral proteincomprising the base sequence.

Exemplary nucleic acid molecules that can modulate protein functioninclude antisense oligonucleotides and RNA interference molecules (e.g.,small interfering RNA (siRNA), microRNA (miRNA) and shRNA).

Antisense oligonucleotides are capable of blocking or decreasing theexpression of a desired target gene by targeting nucleic acids encodingthe gene or subunit thereof. Methods are known to those of ordinaryskill in the art for the preparation of antisense oligonucleotidemolecules that will specifically bind one or more target gene(s) withoutcross-reacting with other polynucleotides. Exemplary sites of targetinginclude, but are not limited to, the initiation codon, the 5′ regulatoryregions, including promoters or enhancers, the coding sequence,including any conserved consensus regions, and the 3′ untranslatedregion. In some embodiments, the antisense oligonucleotides are about 10to about 100 nucleotides in length, about 15 to about 50 nucleotides inlength, about 18 to about 25 nucleotides in length, or more. In certainembodiments, the oligonucleotides further comprise chemicalmodifications to increase nuclease resistance and the like, such as, forexample, phosphorothioate linkages and 2′-O-sugarmodifications known tothose of ordinary skill in the art.

RNA interference (RNAi) is a biological process in which RNA moleculesinhibit gene expression or translation by neutralizing targeted mRNAmolecules. Specifically, RNAi refers to a post-transcriptional silencingmechanism initiated by small double-stranded RNA molecules that suppressexpression of genes with sequence homology. Key to the mechanism of RNAiare small interfering RNA (siRNA) strands, which have complementarynucleotide sequences to a targeted messenger RNA (mRNA) molecule. siRNAsare short, single-stranded nucleic acid molecules capable of inhibitingor down-regulating gene expression in a sequence-specific manner; see,for example, Zamore et al., Cell 101:25 33 (2000); Bass, Nature411:428-429(2001); Elbashir et al., Nature 411:494-498 (2001); andKreutzer et al., International PCT Publication No. WO 00/44895;Zernicka-Goetz et al., International PCT Publication No. WO 01/36646;Fire, International PCT Publication No. WO 99/32619; Plaetinck et al.,International PCT Publication No. WO 00/01846; Mello and Fire,International PCT Publication No. WO 01/29058; Deschamps-Depaillette,International PCT Publication No. WO 99/07409; and Li et al.,International PCT Publication No. WO 00/44914. Methods of preparing asiRNA molecule for use in gene silencing are described in U.S. Pat. No.7,078,196, which is hereby incorporated by reference. Generally, onewould prepare siRNA molecules that will specifically target one or moremRNAs without cross-reacting with other polynucleotides. siRNA moleculescan be generated by methods known in the art, such as by typical solidphase oligonucleotide synthesis, and often will incorporate chemicalmodifications to increase half-life and/or efficacy of the siRNA agent,and/or to allow for a more robust delivery formulation. Alternatively,siRNA molecules are delivered using a vector encoding an expressioncassette for intracellular transcription of siRNA.

Nucleic Acids Encoding Influenza Polypeptides

In some aspects, the polypeptides described herein are encoded by anucleic acid molecule (e.g., DNA, RNA).

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invivo when placed under the control of appropriate regulatory sequences(or “control elements”). The boundaries of the coding sequence aredetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A coding sequence can include,but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA,genomic DNA sequences from viral or procaryotic DNA, and even syntheticDNA sequences. A transcription termination sequence may be located 3′ tothe coding sequence. Transcription and translation of coding sequencesare typically regulated by “control elements,” including, but notlimited to, transcription promoters, transcription enhancer elements,transcription termination signals, polyadenylation sequences (located 3′to the translation stop codon), sequences for optimization of initiationof translation (located 5′ to the coding sequence), and translationtermination sequences.

A “promoter” is a nucleotide sequence which initiates transcription of apolypeptide-encoding polynucleotide. Promoters can include induciblepromoters (where expression of a polynucleotide sequence operably linkedto the promoter is induced by an analyte, cofactor, regulatory protein,etc.), repressible promoters (where expression of a polynucleotidesequence operably linked to the promoter is repressed by an analyte,cofactor, regulatory protein, etc.), and constitutive promoters. Inaddition, such promoters can also have tissue specificity, for example,the CD80 promoter is only inducible in certain immune cells, and themyoD promoter is only inducible in muscle cells. It is intended that theterm “promoter” or “control element” includes full-length promoterregions and functional (e.g., controls transcription or translation)segments of these regions. A promoter is “derived from” a gene encodinga co-stimulatory molecule if it has the same or substantially the samebasepair sequence as a region of the promoter region of theco-stimulatory molecule, complements thereof, or if it displays sequenceidentity as described below.

A “vector” is capable of transferring gene sequences to target cells(e.g., viral vectors, non-viral vectors, particulate carriers, andliposomes). Typically, “vector construct,” “expression vector,” and“gene transfer vector,” mean any nucleic acid construct capable ofdirecting the expression of a gene of interest and which can transfergene sequences to target cells. Thus, the term includes cloning andexpression vehicles, as well as viral vectors.

Nucleotide sequences selected for use in the present disclosure can bederived from known sources, for example, by isolating the same fromcells containing a desired gene or nucleotide sequence using standardtechniques. Similarly, the nucleotide sequences can be generatedsynthetically using standard modes of polynucleotide synthesis that arewell known in the art. See, e.g., Edge et al. (1981) Nature 292:756-762;Nambair et al. (1994) Science 223:1299-1301: Jay et al. (1984) J. Biol.Chem. 259:6311-6317. Generally, synthetic oligonucleotides can beprepared by either the phosphotriester method as described by Edge etal., supra, and Duckworth et al. (1981) Nucleic Acids Res. 9:1691-1706,or the phosphoramidite method as described by Beaucage et al. (1981)Tet. Letts. 22:1859, and Matteucci et al. (1981) J. Am. Chem.Soc.103:3185.

Another method for obtaining nucleic acid sequences for use herein is byrecombinant means. Thus, a desired nucleotide sequence can be excisedfrom a plasmid carrying the same using standard restriction enzymes andprocedures. Site specific DNA cleavage is performed by treating with thesuitable restriction enzyme (or enzymes) under conditions which aregenerally understood in the art, and the particulars of which arespecified by manufacturers of commercially available restrictionenzymes. If desired, size separation of the cleaved fragments may beperformed by polyacrylamide gel or agarose gel electrophoresis usingstandard techniques.

Yet another convenient method for isolating specific nucleic acidmolecules is by the polymerase chain reaction (PCR). Mullis et al.(1987) Methods Enzymol. 155:335-350. This technique uses DNA polymerase,usually a thermostable DNA polymerase, to replicate a desired region ofDNA. The region of DNA to be replicated is identified byoligonucleotides of specified sequence complementary to opposite endsand opposite strands of the desired DNA to prime the replicationreaction. The product of the first round of replication is itself atemplate for subsequent replication, thus repeated successive cycles ofreplication result in geometric amplification of the DNA fragmentdelimited by the primer pair used. This method also allows for thefacile addition of nucleotide sequences onto the ends of the DNA productby incorporating these added sequences onto the oligonucleotide primers(see, e.g., PCR Protocols, A Guide to Methods and Applications, Innis etal (eds) Harcourt Brace Jovanovich Publishers, NY (1994)). PCRconditions used for each amplification reaction are empiricallydetermined. A number of parameters influence the success of a reaction.Among them are annealing temperature and time, extension time, Mg2+ andATP concentration, pH, and the relative concentration of primers,templates, and deoxyribonucleotides.

Once coding sequences for desired proteins have been prepared orisolated, such sequences can be cloned into any suitable vector orreplicon. Numerous cloning vectors are known to those of skill in theart, and the selection of an appropriate cloning vector is a matter ofchoice. Ligations to other sequences are performed using standardprocedures, known in the art.

In some aspects, a nucleic acid molecule described herein is provided inan expression vector. In some embodiments, the vector comprises thenucleic acid molecule that codes for the peptides operatively linked toappropriate expression control sequences. Methods of affecting thisoperative linking, either before or after the nucleic acid molecule isinserted into the vector, are well known. Expression control sequencesinclude promoters, activators, enhancers, operators, ribosomal nucleasedomains, start signals, stop signals, cap signals, polyadenylationsignals, and other signals involved with the control of transcription ortranslation.

Viral vectors that are suitable for use include, for example,retroviral, adenoviral, and adeno-associated vectors, herpes virus,simian virus 40 (SV40), and bovine papilloma virus vectors (see, forexample, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press,Cold Spring Harbor, N.Y.).

A number of viral based systems have been used for gene delivery. Forexample, retroviral systems are known and generally employ packaginglines which have an integrated defective provirus (the “helper”) thatexpresses all of the genes of the virus but cannot package its owngenome due to a deletion of the packaging signal, known as the psisequence. Thus, the cell line produces empty viral shells. Producerlines can be derived from the packaging lines which, in addition to thehelper, contain a viral vector which includes sequences required in cisfor replication and packaging of the virus, known as the long terminalrepeats (LTRs). The gene of interest can be inserted in the vector andpackaged in the viral shells synthesized by the retroviral helper. Therecombinant virus can then be isolated and delivered to a subject. (See,e.g., U.S. Pat. No. 5,219,740.) Representative retroviral vectorsinclude but are not limited to vectors such as the LHL, N2, LNSAL, LSHLand LHL2 vectors described in e.g., U.S. Pat. No. 5,219,740,incorporated herein by reference in its entirety, as well as derivativesof these vectors, such as the modified N2 vector described herein.Retroviral vectors can be constructed using techniques well known in theart. See, e.g., U.S. Pat. No. 5,219,740; Mann et al. (1983) Cell33:153-159.

Adenovirus based systems have been developed for gene delivery and aresuitable for delivering the nucleic acid molecules described herein.Human adenoviruses are double-stranded DNA viruses which enter cells byreceptor-mediated endocytosis. These viruses are particularly wellsuited for gene transfer because they are easy to grow and manipulateand they exhibit a broad host range in vivo and in vitro. For example,adenoviruses can infect human cells of hematopoietic, lymphoid andmyeloid origin. Furthermore, adenoviruses infect quiescent as well asreplicating target cells. Unlike retroviruses which integrate into thehost genome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis. The virus is easilyproduced at high titers and is stable so that it can be purified andstored. Even in the replication-competent form, adenoviruses cause onlylow level morbidity and are not associated with human malignancies.Accordingly, adenovirus vectors have been developed which make use ofthese advantages. For a description of adenovirus vectors and their usessee, e.g., Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al.(1993) J. Virol. 67:5911-5921; Mittereder et al. (1994) Human GeneTherapy 5:717-729; Seth et al. (1994) J. Virol. 68:933-940; Barr et al.(1994) Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques6:616-629; Rich et al. (1993) Human Gene Therapy 4:461-476.Adeno-associated viral vector (AAV) can also be used to administer thepolynucleotides described herein. AAV vectors can be derived from anyAAV serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAVX7, etc. AAV vectors can have one or more of the AAV wild-typegenes deleted in whole or part, preferably the rep and/or cap genes, butretain one or more functional flanking inverted terminal repeat (ITR)sequences. Functional ITR sequences are necessary for the rescue,replication and packaging of the AAV virion. Thus, an AAV vectorincludes at least those sequences required in cis for replication andpackaging (e.g., functional ITRs) of the virus. The ITR sequence neednot be the wild-type nucleotide sequence, and may be altered, e.g., bythe insertion, deletion or substitution of nucleotides, so long as thesequence provides for functional rescue, replication and packaging.

AAV expression vectors are constructed using known techniques to atleast provide as operatively linked components in the direction oftranscription, control elements including a transcriptional initiationregion, the DNA of interest and a transcriptional termination region.The control elements are selected to be functional in a mammalian cell.The resulting construct which contains the operatively linked componentsis bounded (5′ and 3′) with functional AAV ITR sequences. Suitable AAVconstructs can be designed using techniques well known in the art. See,e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International PublicationNos. WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4Mar. 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

Models for Assessing Prophylactic and Therapeutic Efficacy

In Vitro Models

In some embodiments, in vitro evaluation are utilized to screen vaccinecandidates. See e.g., Tapia-Calle, G., et al., Vaccines (Basel) 5(3)pii: E21 (2017). doi: 10.3390/vaccines5030021. Dendritic cells (DCs)play an important in the development of innate and adaptive immuneresponses. In a study, a DC line (MUT-3) and primary monocyte-derivedDCs (Mo-DCs) were employed to screen whole inactivated and subunitinfluenza vaccines. The Mo-DCs were stimulated with both vaccines andshowed upregulated protein expression of activation markers (MHC II,CD86 and CD40) and changes in cytokine secretion in response to wholeinactivated vaccines. The Mo-DCs additionally showed increase in genecoding for surface markers of DC cells. The results show that Mo-DCsderived from either fresh or frozen/thawed PBMCs could be utilized toscreen vaccine candidates.

In another embodiment, long-term cultures of unfractionated PBMCS wereemployed to assess recall T cell responses to vaccine candidates. Seee.g., Tapia-Calle, G., et al., Vaccines (Basel) 7(4). pii: E181 (2019).doi: 10.3390/vaccines7040181. After stimulation with whole inactivatedand subunit influenza vaccines. T cell-mediated immune responses, e.g.,activation, proliferation, increase in cytotoxic potential and IFN-γresponses were evaluated. CD4+ and CD8+ phenotyping showed that effectorand central memory T cells were activated. Additionally, vaccine inducedfollicular T helper cell responses (T_(FH)) were also elicited.

In some embodiments, long-term cultures human precision-cut lung slices(PCLS) from human donors are used as an ex vivo model to evaluate immuneresponse to stimulation by influenza vaccine. See e.g., Temann, A., etal., Hum Vac Immunother 13(2):351-358 (2017). Upon stimulation withinfluenza vaccines, PCLS showed upregulation of cytokine secretions,e.g., IFN-γ, TNF-α and IL-2.

In Vivo Models

Various animal models for evaluating influenza vaccines are known in theart. Margine, I., Krammer, F., Pathogens 3(4):845-874 (2014).Immunogenicity and protective efficacy of candidate influenza vaccineshave been tested in e.g., chicken, mouse, ferret, pigs, and non-humanprimates models.

Ferrets were the first species to be successfully infected with humaninfluenza isolates, and is susceptible to a wide range of human isolateswithout prior adaptation. Ferrets display clinical symptoms similar tohuman disease when infected with human influenza, although the presenceand severity of symptoms vary depending on the challenge viral strainand route of administration.

Wild mice are not natural hosts of the influenza viruses. However, miceare widely used in influenza research due to their small size, low cost,availability of immunological reagents, availability of laboratory micestrains that can be infected with certain influenza, and availability oftransgenic mice strains with targeted gene disruptions to study hostresponses. Generally, influenza viruses require adaption in mice to beable to infect mice and replicate. The process of adaptation, i.e.,repeated in vivo passage in mouse lungs will cause antigenic andphenotypic changes in the adapted virus. However, several pathogenicpandemic influenza strains, such as H1N1, H5N1, and H7, are able tocause disease in mice without prior adaption.

Pigs are an attractive model for influenza research as they arenaturally infected by both human and avian influenza viruses. Innate andadaptive B- and T-cell immunity against influenza have beencharacterized in the pig model. Holzer, B., et al., Front. Immunol.10:98 (2019). doi: 10.3389/fimmu.2019.00098.

Immunogenicity and challenge influenza studies have been conducted inpigs. For example cold adapted 2017-2018 Northern Hemisphere LAIVvaccine Fluenz Tetra (AstraZeneca) containing two type A viruses: H1N1A/Slovenia/2903/2015, MEDI 279432 107.0±0.5 FFU [A/Michigan/45/2015(H1N1) pdm09-like strain]; H3N2 A/New Caledonia/71/2014, MEDI 263122107.0±0.5 FFU [A/Hong Kong/4801/2014 (H3N2)-like strain] and two type B(IBV) viruses; (B/Brisbane/60/2008, MEDI 228030) 107.0±0.5 FFU(B/Brisbane/60/2008-like strain) and B/Phuket/3073/2013, MEDI 254977)107.0±0.5 FFU (B/Phuket/3073/2013-like strain) were administeredintranasally to pigs. Holzer, B., et al., Front. Immunol 10:2625 (2019).doi: 10.3389/fimmu.2019.02625. Four weeks after immunization, the pigswere challenged intranasally with wild-type viruses contained in theLAIV vaccine.

Nasal swabs were collected to test virus load. Serum and bronchoalveolarlavage (BAL) fluid were collected and tested for antibody andneutralizing antibody titers in ELISA and microneutralization (MN)assays, respectively. Cellular response were tested in IFN-γ ELISPOT andintracellular cytokine staining of cells collected from peripheralblood, trachea bronchial lymph nodes (TBLNs) and BALs.

Nonhuman primates are naturally infected by human influenza virus, andare considered good models of human responses to influenza infection andvaccination. Although ethical and economical considerations limit theuse of non-human primates in influenza vaccine research, their use ischallenge experiments are useful in testing pandemic influenza virusstrains.

In some embodiments, the immunogenic compositions herein are tested inimmunogenicity and/or challenge studies in animal models.

Immunogenic Compositions

Also provided herein are immunogenic compositions (e.g., vaccines)comprising combinations or cocktails of the recombinant viral proteinsand/or polypeptides described herein. In some embodiments, theimmunogenic compositions comprise a nucleic acid molecule encoding therecombinant viral proteins and/or polypeptides described herein. In someembodiments, the compositions further comprise a pharmaceuticallyacceptable carrier.

In some embodiments, immunogenic compositions described herein furthercomprise one or more adjuvants. For example, alum, aluminum salts(Baylor et al., 2002, Vaccine, 20:S18; incorporated herein by reference)and monophosphoryl lipid A (MPL; Ribi et al., 1986, Immunology andImmunopharmacology of Bacterial Endotoxins, Plenum Publ. Corp., NY, p.407; incorporated herein by reference) can be used as adjuvants in humanvaccines. Alternatively or additionally, new compounds are currentlybeing tested as adjuvants in human vaccines, such as: MF59 (See, e.g.,Ott et al., “MF59—Design and Evaluation of a Safe and Potent Adjuvantfor Human Vaccines” in Vaccine Design: The Subunit and Adjuvant Approach(Powell, M. F. and Newman, M. J. eds.) Plenum Press, New York, 1995, pp.277-296; incorporated herein by reference); CpG oligodeoxynudeotide(ODN) adjuvants such as CPG 7909 (Cooper et al., 2004, Vaccine, 22:3136;incorporated herein by reference); Monophosphoryl lipid A (MPL)adjuvants and lipid A mimetis including AS04 (Didierlaurent, A. M. etal, J. Immunol., 2009, 183: 6186-6197; incorporated by referenceherein), monophosphoryl lipid A (MPL, GSK) and glucopyranosyl lipid AGLA (Immune Design Corporation, IDC); AF03 (Klucker, M. F. et al, J.Pharm Sci., 2012, 101: 4490-4500; incorporated herein by reference); theTLR-3 ligand polyinosinic:polycytidylic acid [poly(I:C)]; TLR9 adjuvantssuch as IC31 (Riedl, K. et al., Vaccine, 2008, 26: 3461-3468;incorporated herein by reference); imidazoquinolines (double cyclicorganic molecules that act as TLR-7/8 agonists) such as imiquimod (R837)or resiquimod (R848); saponins such as QS21 (Ghochikyan et al., 2006,Vaccine, 24:2275; incorporated herein by reference), ISCOMATRIX adjuvant(Duewell, P., et al., J. Immunol, 2011, 187: 55-63; incorporated hereinby reference), and Matrix-M™ (Novavax).

Additionally, some adjuvants are known in the art to enhance theimmunogenicity of influenza vaccines, such aspoly[di(carboxylatophenoxy)phosphazene] (PCCP; Payne et al., 1998,Vaccine, 16:92; incorporated herein by reference), interferon-.gamma.(Cao et al., 1992, Vaccine, 10:238; incorporated herein by reference),block copolymer P1205 (CRL1005; Katz et al., 2000, Vaccine, 18:2177;incorporated herein by reference), interleukin-2 (IL-2; Mbwuike et al.,1990, Vaccine, 8:347; incorporated herein by reference), and polymethylmethacrylate (PMMA; Kreuter et al., 1981, J. Pharm. Sci., 70:367;incorporated herein by reference).

In some embodiments, the immunogenic compositions include one or moreinactive agents such as a sterile, biocompatible carrier including, butnot limited to, sterile water, saline, buffered saline, or dextrosesolution. In some embodiments, the composition contains any of a varietyof additives, such as stabilizers, buffers, excipients (e.g., sugars,amino acids, etc.), or preservatives. Pharmaceutically acceptablecarriers used in particular embodiments include, but are not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. In some embodiments, the carrier and compositionare sterile, and the formulation suits the mode of administration. Insome embodiments, an immunogenic composition contains minor amounts ofwetting or emulsifying agents, or pH buffering agents. In someembodiments, a pharmaceutical composition is a liquid solution,suspension, emulsion, tablet, pill, capsule, sustained releaseformulation, or powder. Oral formulations can include standard carrierssuch as pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, and magnesium carbonate. Any ofthe common pharmaceutical carriers, such as sterile saline solution orsesame oil, can be used. The medium can also contain conventionalpharmaceutical adjunct materials such as, for example, pharmaceuticallyacceptable salts to adjust the osmotic pressure, buffers, preservativesand the like. Other media that can be used with the compositions andmethods provided herein are normal saline and sesame oil.

In some embodiments, an immunogenic composition is formulated forintradermal injection, intranasal administration or intramuscularinjection. In some embodiments, injectables are prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. In some embodiments, injection solutions and suspensions areprepared from sterile powders, granules, and. General considerations inthe formulation and manufacture of pharmaceutical agents foradministration by these routes may be found, for example, in Remington'sPharmaceutical Sciences, 19.sup.th ed., Mack Publishing Co., Easton,Pa., 1995; incorporated herein by reference. At present the oral ornasal spray or aerosol route (e.g., by inhalation) are most commonlyused to deliver therapeutic agents directly to the lungs and respiratorysystem. In some embodiments, compositions in accordance with theinvention are administered using a device that delivers a metered dosageof composition (e.g., of an optimized HA polypeptide). Suitable devicesfor use in delivering intradermal pharmaceutical compositions describedherein include short needle devices such as those described in U.S. Pat.Nos. 4,886,499, 5,190,521, 5,328,483, 5,527,288, 4,270,537, 5,015,235,5,141,496, 5,417,662 (all of which are incorporated herein byreference).

Intradermal compositions may also be administered by devices which limitthe effective penetration length of a needle into the skin, such asthose described in WO1999/34850, incorporated herein by reference, andfunctional equivalents thereof. Also suitable are jet injection deviceswhich deliver liquid vaccines to the dermis via a liquid jet injector orvia a needle which pierces the stratum corneum and produces a jet whichreaches the dermis. Jet injection devices are described for example inU.S. Pat. Nos. 5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912,5,569,189, 5,704,911, 5,383,851, 5,893,397, 5,466,220, 5,339,163,5,312,335, 5,503,627, 5,064,413, 5,520,639, 4,596,556, 4,790,824,4,941,880, 4,940,460, WO1997/37705, and WO1997/13537 (all of which areincorporated herein by reference). Also suitable are ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis. Additionally, conventional syringes may be used in the classicalmantoux method of intradermal administration.

Preparations for parenteral administration include sterile aqueous ornonaqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

In some embodiments, the compositions are administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Methods of Use

In some embodiments, the polypeptides described herein are capable ofeliciting an immune response against an influenza virus. In someembodiments, the polypeptides can be used as vaccines to protectindividuals against influenza infection. In some embodiments, thenucleic acid molecules encoding polypeptides described herein arecapable of eliciting an immune response against an influenza virus. Insome embodiments, the nucleic acid molecules can be used as vaccines toprotect individuals against influenza infection.

In some embodiments, the disclosure provides a method of vaccinating asubject against influenza, in particular, against various strains ofinfluenza. Such methods employ the immunogenic compositions of thepresent disclosure. Accordingly, in some embodiments, the methodcomprises administering an immunogenic composition to a subject suchthat an immune response against influenza virus is produced in thesubject. In some embodiments, the polypeptides described herein arecapable of eliciting neutralizing antibodies to influenza. In someembodiments, the nucleic acid molecules encoding polypeptides describedherein are capable of eliciting neutralizing antibodies to influenza.

Immunogenic compositions of the present disclosure can be used tovaccinate individuals using a prime/boost protocol. Such a protocol isdescribed in U.S. Patent Publication No. 2011/0177122, which isincorporated herein by reference in its entirety. In such a protocol, afirst immunogenic composition may be administered to the individual(prime) and then after a period of time, a second immunogeniccomposition may be administered to the individual (boost).Administration of the boosting composition is generally weeks or monthsafter administration of the priming composition, preferably about 2-3weeks or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or28 weeks, or 32 weeks. In one embodiment, the boosting composition isformulated for administration about 1 week, or 2 weeks, or 3 weeks, or 4weeks, or 5 weeks, or 6 weeks, or 7 weeks, or 8 weeks, or 9 weeks, or 16weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks afteradministration of the priming composition.

In some embodiments, the subject is at risk for infection withinfluenza. In some embodiments, the subject has been exposed toinfluenza. For example, the subject may be an elderly individual, achild, an infant or an immunocompromised individual. As used herein, theterms exposed, exposure, and the like, indicate the subject has come incontact with a person or animal that is known to be infected withinfluenza. Immunogenic compositions of the present disclosure may beadministered using techniques well known to those in the art anddescribed herein.

In some embodiments, the polypeptides and immunogenic compositions ofthe present disclosure is used to protect a subject against infection byantigenically divergent influenza. In some embodiments, the nucleic acidmolecules and immunogenic compositions of the present disclosure is usedto protect a subject against infection by antigenically divergentinfluenza.

Methods of preparing and administering immunogenic compositions to asubject in need thereof are well known in the art or readily determinedby those skilled in the art. The dosage and frequency of administrationmay depend on whether the treatment is prophylactic or therapeutic.

The immunogenic composition and polypeptides of the disclosure aresuitable for administration to mammals (e.g., primates, (e.g., humans,chimpanzees, monkeys, baboons), rats (e.g., cotton rats), mice, cows(e.g., calves), guinea pigs, ferrets and hamsters). In some embodiments,the disclosure provides a method of inducing an immune response in amammal, comprising the step of administering a composition (e.g., animmunogenic composition) of the disclosure to the mammal. Thecompositions (e.g., an immunogenic composition) can be used to produce avaccine formulation for immunizing a mammal. The mammal is typically ahuman, and the immunogenic composition typically comprises a polypeptidecomprising an amino acid sequence of an influenza viral protein. In someembodiments, the mammal is a human, and the immunogenic compositioncomprises a nucleic acid molecule encoding a polypeptide comprising anamino acid sequence of an influenza viral protein.

The disclosure also provides a composition of for use as a medicament,e.g., for use in immunizing a patient against influenza infection.

The disclosure also provides the use of a polypeptide as described abovein the manufacture of a medicament for raising an immune response in apatient. In some embodiments, the disclosure provides the use of anucleic acid molecule described herein in the manufacture of amedicament for raising an immune response in a patient.

The immune response raised by these methods and uses will generallyinclude an antibody response, preferably a protective antibody response.Methods for assessing antibody responses after influenza vaccination arewell known in the art.

Compositions of the invention can be administered in a number ofsuitable ways, such as intramuscular injection (e.g., into the arm orleg), subcutaneous injection, intranasal administration, oraladministration, intradermal administration, transcutaneousadministration, transdermal administration, and the like. Theappropriate route of administration will be dependent upon the age,health and other characteristics of the mammal A clinician will be ableto determine an appropriate route of administration based on these andother factors.

Immunogenic compositions, and vaccine formulations, may be used to treatboth children and adults, including pregnant women. Thus a subject maybe less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old,or at least 55 years old. Preferred subjects for receiving the vaccinesare the elderly (e.g., >50 years old, >60 years old, >65 years, andpreferably >75 years), the young (e.g., <6 years old, such as 4-6 yearsold, <5 years old), and pregnant women. The vaccines are not limited tothese groups, however, and may be used more generally in a population.

Treatment can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunization schedule and/or ina booster immunization schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes, e.g., a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Administration of more than one dose (typically two doses) isparticularly useful in immunologically naive patients. Multiple doseswill typically be administered at least 1 week apart (e.g., about 2weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about10 weeks, about 12 weeks, about 16 weeks, and the like.)

Vaccine formulations produced using a composition of the disclosure maybe administered to patients at substantially the same time as (e.g.,during the same medical consultation or visit to a healthcareprofessional or vaccination center) other vaccines.

In some embodiments, the immunogenic compositions, polypeptides ornucleic acid molecules described herein are administered as atherapeutic to a subject infected with influenza.

Kits

The immunogenic composition or polypeptide of the disclosure can beprovided in a kit. In some embodiments, a nucleic acid molecule of thedisclosure is provided in a kit. In some embodiments, the kit includes(a) a container that contains a composition that includes one or moreunit doses of the immunogenic composition or polypeptide, and optionally(b) instructions for use. In some embodiments, the kit includes (a) acontainer that contains a composition that includes one or more unitdoses of the immunogenic composition or nucleic acid molecule, andoptionally (b) instructions for use. The unit doses of the immunogeniccomposition or polypeptide are sufficient to cause an immunogenicresponse (e.g., antibody production) in a subject. In some embodiments,the unit doses of the immunogenic composition or nucleic acid moleculeare sufficient to cause an immunogenic response (e.g., antibodyproduction) in a subject. The kit can also include reagents andinstructions useful in the testing (assaying) for an immunogenicresponse. Such methods of assaying for an immunogenic response include,but are not limited to, any of the testing methods described herein. Inone embodiment, the kit includes one or more additional agents fortreating influenza. For example, the kit includes a first container thatcontains a composition that includes the immunogenic composition, and asecond container that includes the one or more additional agents.

In some embodiments, the instructions provide methods of administeringthe immunogenic composition, e.g., in a suitable dose, dosage form, ormode of administration (e.g., a dose, dosage form, or mode ofadministration described herein), to treat a subject who is infectedwith influenza, or who is at risk of being infected with influenza.

In addition to the immunogenic composition or polypeptide, thecomposition in the kit can include other ingredients, such as a solventor buffer, a stabilizer, or a preservative. The agent can be provided inany form, e.g., liquid, dried or lyophilized form, preferablysubstantially pure and/or sterile. When the agents are provided in aliquid solution, the liquid solution preferably is an aqueous solution.When the agents are provided as a dried form, reconstitution generallyis by the addition of a suitable solvent. The solvent, e.g., sterilewater or buffer, can optionally be provided in the kit.

The kit can include one or more containers for the composition orcompositions containing the agents. In some embodiments, the kitcontains separate containers, dividers or compartments for thecomposition and informational material. For example, the composition canbe contained in a bottle, vial, or syringe, and the informationalmaterial can be contained in a plastic sleeve or packet. In otherembodiments, the separate elements of the kit are contained within asingle, undivided container. For example, the composition is containedin a bottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some embodiments, the kit includes aplurality (e.g., a pack) of individual containers, each containing oneor more unit dosage forms (e.g., a dosage form described herein) of theagents. The containers can include a combination unit dosage, e.g., aunit that includes both the polypeptide and the second agent, e.g., in adesired ratio. For example, the kit includes a plurality of syringes,ampules, foil packets, blister packs, or medical devices, e.g., eachcontaining a single combination unit dose. The containers of the kitscan be air tight, waterproof (e.g., impermeable to changes in moistureor evaporation), and/or light-tight.

The kit optionally includes a device suitable for administration of thecomposition, e.g., a syringe or other suitable delivery device. Thedevice can be provided pre-loaded with one or both of the agents or canbe empty, but suitable for loading.

Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Further, unless otherwiserequired by context, singular terms shall include pluralities and pluralterms shall include the singular.

As used herein, “about” will be understood by persons of ordinary skilland will vary to some extent depending on the context in which it isused. If there are uses of the term which are not clear to persons ofordinary skill given the context in which it is used, “about” will meanup to plus or minus 10% of the particular value.

As used herein, the term “alanine scanning” refers to a technique usedto determine the contribution of a specific wild-type residue to thestability or function(s) (e.g., binding affinity) of a given protein orpolypeptide. The technique involves the substitution of an alanineresidue for a wild-type residue in a polypeptide, followed by anassessment of the stability or function(s) (e.g., binding affinity) ofthe alanine-substituted derivative or mutant polypeptide and comparisonto the wild-type polypeptide. Techniques to substitute alanine for awild-type residue in a polypeptide are known in the art.

The term “ameliorating” refers to any therapeutically beneficial resultin the treatment of a disease state, e.g., infection, lessening in theseverity or progression, remission, or cure thereof.

As used herein, the term “amino acid” refers to naturally occurring andsynthetic amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., a carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that function in amanner similar to a naturally occurring amino acid.

Amino acids can be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,can be referred to by their commonly accepted single-letter codes.

As used herein, an “amino acid substitution” refers to the replacementof at least one existing amino acid residue in a predetermined aminoacid sequence (an amino acid sequence of a starting polypeptide) with asecond, different “replacement” amino acid residue. An “amino acidinsertion” refers to the incorporation of at least one additional aminoacid into a predetermined amino acid sequence. While the insertion willusually consist of the insertion of one or two amino acid residues,larger “peptide insertions,” can also be made, e.g., insertion of aboutthree to about five or even up to about ten, fifteen, or twenty aminoacid residues. The inserted residue(s) can be naturally occurring ornon-naturally occurring as disclosed above. An “amino acid deletion”refers to the removal of at least one amino acid residue from apredetermined amino acid sequence. The following table providesexemplary and preferred substitutions for all 20 amino acids.

Original Exemplary Preferred Residues Substitutions Substitutions AlaVal, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser,Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, ArgArg Ile Leu, Val, Met, Ala, Phe, Leu Norleucine Leu Norleucine, Ile,Val, Met, Ile Ala, Phe Lys Arg, 1,4 Diamino-butyric Arg acid, Gln, AsnMet Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu Pro Ala Gly SerThr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, SerPhe Val Ile, met, Leu, Phe, Ala, Leu norleucine

The term “antigen presenting cell” or “APC” is a cell that displaysforeign antigen complexed with MHC on its surface. T cells recognizethis complex using T cell receptor (TCR). Examples of APCs include, butare not limited to, dendritic cells (DCs), peripheral blood mononuclearcells (PBMC), monocytes (such as THP-1), B lymphoblastoid cells (such asC1R.A2, 1518 B-LCL) and monocyte-derived dendritic cells (DCs). SomeAPCs internalize antigens either by phagocytosis or by receptor-mediatedendocytosis.

The term “antigen presentation” refers to the process by which APCscapture antigens and enables their recognition by T cells, e.g., as acomponent of an MHC-I and/or MHC-II conjugate.

As used herein, the term “base pair” refers to two nucleobases onopposite complementary nucleic acid strands that interact via theformation of specific hydrogen bonds. As used herein, the term“Watson-Crick base pairing”, used interchangeably with “complementarybase pairing”, refers to a set of base pairing rules, wherein a purinealways binds with a pyrimidine such that the nucleobase adenine (A)forms a complementary base pair with thymine (T) and guanine (G) forms acomplementary base pair with cytosine (C) in DNA molecules. In RNAmolecules, thymine is replaced by uracil (U), which, similar to thymine(T), forms a complementary base pair with adenine (A). The complementarybase pairs are bound together by hydrogen bonds and the number ofhydrogen bonds differs between base pairs. As in known in the art,guanine (G)-cytosine (C) base pairs are bound by three (3) hydrogenbonds and adenine (A)-thymine (T) or uracil (U) base pairs are bound bytwo (2) hydrogen bonds. Base pairing interactions that do not followthese rules can occur in natural, non-natural, and synthetic nucleicacids and are referred to herein as “non-Watson-Crick base pairing” oralternatively “non-complementary base pairing”.

A polypeptide or amino acid sequence “derived from” a designatedpolypeptide or protein refers to the origin of the polypeptide.Preferably, the polypeptide or amino acid sequence which is derived froma particular sequence has an amino acid sequence that is essentiallyidentical to that sequence or a portion thereof, wherein the portionconsists of at least 10-20 amino acids, preferably at least 20-30 aminoacids, more preferably at least 30-50 amino acids, or which is otherwiseidentifiable to one of ordinary skill in the art as having its origin inthe sequence. Polypeptides derived from another peptide can have one ormore mutations relative to the starting polypeptide, e.g., one or moreamino acid residues which have been substituted with another amino acidresidue or which has one or more amino acid residue insertions ordeletions.

A polypeptide can comprise an amino acid sequence which is not naturallyoccurring. Such variants necessarily have less than 100% sequenceidentity or similarity with the starting molecule. In certainembodiments, the variant has an amino acid sequence from about 75% toless than 100% amino acid sequence identity or similarity with the aminoacid sequence of the starting polypeptide, more preferably from about80% to less than 100%, more preferably from about 85% to less than 100%,more preferably from about 90% to less than 100% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% to lessthan 100%, e.g., over the length of the variant molecule.

In some embodiments, there is one amino acid difference between astarting polypeptide sequence and the sequence derived there from.Identity or similarity with respect to this sequence is defined hereinas the percentage of amino acid residues in the candidate sequence thatare identical (i.e., same residue) with the starting amino acidresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. In certainembodiments, a polypeptide consists of, consists essentially of, orcomprises an amino acid sequence selected from a sequence set forth inthe sequence listing table. In certain embodiments, a polypeptideincludes an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence selected from a sequence set forthin the sequence listing table. In certain embodiments, a polypeptideincludes a contiguous amino acid sequence at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a contiguous amino acid sequence selected froma sequence set forth in the sequence listing table. In certainembodiments, a polypeptide includes an amino acid sequence having atleast 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers)contiguous amino acids of an amino acid sequence selected from asequence set forth in the sequence listing table.

In certain embodiments, the polypeptides of the disclosure are encodedby a nucleotide sequence. Nucleotide sequences of the disclosure can beuseful for a number of applications, including: cloning, gene therapy,protein expression and purification, mutation introduction, DNAvaccination of a host in need thereof, antibody generation for, e.g.,passive immunization, PCR, primer and probe generation, and the like. Incertain embodiments, the nucleotide sequence of the invention comprises,consists of, or consists essentially of, a nucleotide sequence selectedfrom a sequence set forth in the sequence listing table. In certainembodiments, a nucleotide sequence includes a nucleotide sequence atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequenceselected from a sequence set forth in the sequence listing table. Incertain embodiments, a nucleotide sequence includes a contiguousnucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto a contiguous nucleotide sequence selected from a sequence set forthin the sequence listing table. In certain embodiments, a nucleotidesequence includes a nucleotide sequence having at least 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300,400, or 500 (or any integer within these numbers) contiguous nucleotidesof a nucleotide sequence selected from a sequence set forth in thesequence listing table.

It will also be understood by one of ordinary skill in the art that thepolypeptides suitable for use in the compositions and methods disclosedherein can be altered such that they vary in sequence from the naturallyoccurring or native sequences from which they were derived, whileretaining the desirable activity of the native sequences. For example,nucleotide or amino acid substitutions leading to conservativesubstitutions or changes at “non-essential” amino acid residues can bemade. Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis.

The polypeptides suitable for use in the compositions and methodsdisclosed herein can, in some embodiments, comprise conservative aminoacid substitutions at one or more amino acid residues, e.g., atessential or non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart, including basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a nonessential amino acid residue in a polypeptide ispreferably replaced with another amino acid residue from the same sidechain family. In some embodiments, a string of amino acids can bereplaced with a structurally similar string that differs in order and/orcomposition of side chain family members. Alternatively, in someembodiments, mutations can be introduced randomly along all or part of acoding sequence, such as by saturation mutagenesis, and the resultantmutants can be incorporated into polypeptides of the disclosure andscreened for their ability to induce an immune response.

As used herein, the term antigen “cross-presentation” refers topresentation of exogenous protein antigens to T cells via MEW class Iand class II molecules on APCs.

As used herein, the term “cytotoxic T lymphocyte (CTL) response” refersto an immune response induced by cytotoxic T cells. CTL responses aremediated primarily by CD8⁺ T cells.

As used herein, the term “effective dose” or “effective dosage” isdefined as an amount sufficient to achieve or at least partially achievethe desired effect. The term “therapeutically effective dose” is definedas an amount sufficient to cure or at least partially arrest the diseaseand its complications in a patient already suffering from the disease.Amounts effective for this use will depend upon the severity of thedisorder being treated and the general state of the patient's own immunesystem.

As used herein, the term “epitope” or “antigenic determinant” refers toa determinant or site on an antigen (e.g., hemagglutinin) to which anantigen-binding protein (e.g., an immunoglobulin, antibody, orantigen-binding fragment) specifically binds. The epitopes of proteinantigens can be demarcated into “linear epitopes” and “conformationalepitopes”. As used herein, the term “linear epitope” refers to anepitope formed from a contiguous, linear sequence of linked amino acids.Linear epitopes of protein antigens are typically retained upon exposureto chemical denaturants (e.g., acids, bases, solvents, cross-linkingreagents, chaotropic agents, disulfide bond reducing agents) or physicaldenaturants (e.g. thermal heat, radioactivity, or mechanical shear orstress). In some embodiments, an epitope is non-linear, also referred toas an interrupted epitope. As used herein, the term “conformationalepitope” or “non-linear epitope” refers to an epitope formed fromnoncontiguous amino acids juxtaposed by tertiary folding of apolypeptide. Conformational epitopes are typically lost upon treatmentwith denaturants. An epitope typically includes at least 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatialconformation. In some embodiments, an epitope includes fewer than 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6or 5 amino acids in a unique spatial conformation. An epitope that isrecognized by a T cell receptor is generally referred to as a T-cellepitope. An epitope that is recognized by an antibody or a B cellreceptor is generally referred to as a B-cell epitope. Generally, anantibody, or antigen-binding fragment thereof, specific for a particulartarget molecule will preferentially recognize and bind to a specificepitope on the target molecule within a complex mixture of proteinsand/or macromolecules. As used herein, the T and/or B cell epitopescomprises conserved amino acid residues, hypervariable amino acidresidues, or combinations thereof of a viral protein. In otherembodiments, the T and/or B cell epitopes comprises conserved amino acidresidues of the viral proteins.

As used herein, the term “epitope mapping” refers to a process or methodof identifying the binding site, or epitope, of an antibody, orantigen-binding fragment thereof, on its target protein antigen. Epitopemapping methods and techniques are provided herein.

As used herein, the term “fragment” in the context of an amino acidsequence refers to an amino acid sequence comprising a portion ofconsecutive amino acid residues from a parent sequence. In a specificembodiment, the term refers to an amino acid sequence of 8 to 15, 10 to20, 2 to 30, 5 to 30, 10 to 60, 25 to 100, 150 to 300 or moreconsecutive amino acid residues from a parent sequence. In anotherembodiment, the term refers to an amino acid sequence of at least 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 125, 150, 175, or 200 consecutive amino acid residues of a parentsequence.

As used herein, the term “hemagglutinin protein” (or “HA protein’)refers to a protein or polypeptide whose amino acid sequence includes atleast one characteristic sequence of an influenza type A or B HA. A widevariety of HA sequences from influenza isolates are known in the art;indeed, the National Center for Biotechnology Information (NCBI)maintains a database (http://www.ncbi.nlm.nih.gov/genomes/FLU/) that, asof the filing of the present application includes approximately 40,000HA sequences (for type A and B viruses). Those of ordinary skill in theart, referring to this database, can readily identify sequences that arecharacteristic of HA polypeptides generally, and/or of particular HApolypeptides (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12,H13, H14, H15, or H16 polypeptides; or of HAs that mediate infection ofparticular hosts, e.g., human, avian, seal etc.). For example, in someembodiments, an HA polypeptide includes one or more characteristicsequence elements found between about residues 97 and about 185, about324 and about 340, about 96 and about 100, and/or about 130 and about230 of an HA protein found in a natural isolate of an influenza virus.

As used herein, “conserved” or “highly conserved regions” are influenzavirus sequences from different strains, or consensus sequences, whichhave been employed to provide an antigen with broad protectiveproperties. Sequence alignments are relied on to yield a “consensus”sequence, where many genetic sequences are incorporated into a singlesequence. A consensus sequence may thus minimize the genetic distancebetween vaccine strains and viruses and so may elicit morecross-reactive immune responses than an immunogen derived from anysingle influenza virus.

As used herein, the term “hybridization” refers to the process of afirst single-stranded nucleic acid, or a portion, fragment, or regionthereof, annealing to a second single-stranded nucleic acid, or aportion, fragment, or region thereof, either from the same or separatenucleic acid molecules, mediated by Watson-Crick base pairing to form asecondary and/or tertiary structure. Complementary strands of linkednucleobases able to undergo hybridization can be from either the same orseparate nucleic acids. Due to the thermodynamically favorable hydrogenbonding interaction between complementary base pairs, hybridization is afundamental property of complementary nucleic acid sequences. Suchhybridization of nucleic acids, or a portion or fragment thereof, mayoccur with “near” or “substantial” complementarity, as well as withexact complementarity.

As used herein, the term “hypervariable” refers to amino acid residuesand/or protein regions that are abundant and surface exposed, and is aprimary target of the immune response against the standard influenzavaccine. The immune response to influenza is overwhelmingly drivenagainst the hypervariable regions of the virus. Thus, in traditionalinfluenza vaccination or natural infections, the protective immuneresponse is overwhelmingly directed at a limited number of continuouslyevolving, strain-specific, primary antigenic determinants on the surfaceof the influenza proteins, and there is minimal cross reaction with orprotection against other serotypes of influenza.

As used herein, the term “non-hypervariable” or“hypervariable-substitute” refers to an amino acid residue that issubstituted for a hypervariable amino acid residue, wherein thesubstitution eliminates or substantially reduces a strain-specificimmune response (e.g., antibody response) against the region containingthe hypervariable amino acid residue. In some embodiments, thenon-hypervariable residue is one that when substituted for thehypervariable amino acid residue, provides surface epitope with reducedantigenicity. In some embodiments, the non-hypervariable residue isselected from is a nonpolar, aliphatic R group amino acid, e.g.,alanine, glycine, valine, leucine, isoleucine, and methionine. In someembodiments, the non-hypervariable residue is conserved at the sameposition in a plurality of influenza strains.

As used herein, the term “immune response” refers to a response of acell of the immune system, such as a B cell, T cell, dendritic cell,macrophage or polymorphonucleocyte, to a stimulus such as an antigen orvaccine. An immune response can include any cell of the body involved ina host defense response, including for example, an epithelial cell thatsecretes an interferon or a cytokine. An immune response includes, butis not limited to, an innate and/or adaptive immune response. As usedherein, a protective immune response refers to an immune response thatprotects a subject from infection (prevents infection or prevents thedevelopment of disease associated with infection). Methods of measuringimmune responses are well known in the art and include, for example,measuring proliferation and/or activity of lymphocytes (such as B or Tcells), secretion of cytokines or chemokines, inflammation, antibodyproduction and the like.

The terms “inducing an immune response” and “enhancing an immuneresponse” are used interchangeably and refer to the stimulation of animmune response (i.e., either passive or adaptive) to a particularantigen. The term “induce” as used with respect to inducing CDC or ADCCrefer to the stimulation of particular direct cell killing mechanisms.

As used herein, the term “influenza strains” is based upon, e.g., theability of influenza to agglutinate red blood cells (RBCS orerythrocytes). Influenza strains are typically categorized based upontheir immunologic or antigenic profile. An HA1 titer is typicallydefined as the highest dilution of a serum that completely inhibitshemagglutination. See, e.g., Schild, et al., Bull. Wld Hlth Org., 1973,48:269-278, etc. Those of skill in the art will be quite familiar withcategorization and classification of influenza into strains and themethods to do so. Antibodies specific for particular influenza strainscan bind to the virus and, thus, prevent such agglutination. Assaysdetermining strain types based on such inhibition are typically known ashemagglutinin inhibition assays (HI assays or HA1 assays) and arestandard and well known methods in the art to characterize influenzastrains. Of course, those of skill in the art will be familiar withother assays, e.g., ELISA, indirect fluorescent antibody assays,immunohistochemistry, Western blot assays, etc. with which tocharacterize influenza strains and the use of and discussion herein ofHI assays should not be necessarily construed as limiting.

As used herein “influenza types and subtypes” are influenza A and Bvirus typically associated with influenza outbreaks in humanpopulations. The type A viruses are categorized into subtypes based upondifferences within their hemagglutinin and neuraminidase surfaceglycoprotein antigens. Hemagglutinin in type A viruses has 14 knownsubtypes and neuraminidase has 9 known subtypes. In humans, currentlyonly about 3 different hemagglutinin and 2 different neuraminidasesubtypes are known, e.g., H1, H2, H3, N1, and N2. In particular, twomajor subtypes of influenza A have been active in humans, namely, H1N1and H3N2. H1N2, however has recently been of concern.

As used herein, the term “influenza vaccine” refers to an immunogeniccomposition capable of stimulating an immune response, administered forthe prevention, amelioration, or treatment of influenza virus infection.An influenza vaccine may include, for example, attenuated or killedinfluenza virus, virus-like particles (VLPs) and/or antigenicpolypeptides (e.g., the engineered hemagglutinins described herein) orDNA derived from them, or any recombinant versions of such immunogenicmaterials.

As used herein, a subject “in need of prevention,” “in need oftreatment,” or “in need thereof,” refers to one, who by the judgment ofan appropriate medical practitioner (e.g., a doctor, a nurse, or a nursepractitioner in the case of humans; a veterinarian in the case ofnon-human mammals), would reasonably benefit from a given treatment(such as treatment with an immunogenic composition).

The term “in vivo” refers to processes that occur in a living organism.

As used herein, “immunogenic composition” refers to a composition thatcomprises at least one antigen which elicits an immunological responsein the host to which the immunogenic composition is administered. Suchimmunological responses can be a cellular and/or antibody-mediatedimmune response to the immunogenic composition.

As used herein, the terms “linked,” “operably linked,” “fused”, or“fusion”, are used interchangeably. These terms refer to the joiningtogether of two more elements or components or domains, by whatevermeans including chemical conjugation or recombinant means. Methods ofchemical conjugation (e.g., using heterobifunctional crosslinkingagents) are known in the art.

As used herein, “MHC molecules” refers to two types of molecules, MHCclass I and MHC class II. MHC class I molecules present antigen tospecific CD8+ T cells and MHC class II molecules present antigen tospecific CD4+ T cells. Antigens delivered exogenously to APCs areprocessed primarily for association with MHC class II. In contrast,antigens delivered endogenously to APCs are processed primarily forassociation with MHC class I.

As used herein, the terms “NA” and “neuraminidase” refer to anyinfluenza neuraminidase, such as an influenza A neuraminidase, aninfluenza B neuraminidase, or an influenza C neuraminidase. A typicalneuraminidase comprises domains known to those of skill in the artincluding a cytoplasmic domain, a transmembrane domain, a stalk domain,and a globular head domain. As used herein, the terms “neuraminidase”and “NA” encompass neuraminidase polypeptides that are modified bypost-translational processing such as disulfide bond formation,glycosylation (e.g., N-linked glycosylation),

As used herein, the term “naturally-occurring” as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

As used herein, the term “nucleic acid” is used in its broadest senseand encompasses any compound and/or substance that includes a polymer ofnucleotides. These polymers are often referred to as polynucleotides.Exemplary nucleic acids or polynucleotides of the disclosure include,but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids(DNAs), DNA-RNA hybrids, RNAi-inducing agents, RNAi agents, siRNAs,shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs thatinduce triple helix formation, threose nucleic acids (TNAs), glycolnucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids(LNAs, including LNA having a β-D-ribo configuration, α-LNA having anα-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a2′-amino functionalization, and 2′-amino-α-LNA having a 2′-aminofunctionalization) or hybrids thereof. A polynucleotide can also containone or more modified bases or DNA or RNA backbones modified forstability or for other reasons. “Modified” bases include, for example,tritylated bases and unusual bases such as inosine. A variety ofmodifications can be made to DNA and RNA; thus, “polynucleotide”embraces chemically, enzymatically, or metabolically modified forms.

As used herein, the term “nucleoside” refers to a compound containing asugar molecule (e.g., a ribose in RNA or a deoxyribose in DNA), orderivative or analog thereof, covalently linked to a nucleobase (e.g., apurine or pyrimidine), or a derivative or analog thereof (also referredto herein as “nucleobase”), but lacking an internucleoside linking group(e.g., a phosphate group). As used herein, the term “nucleotide” refersto a nucleoside covalently bonded to an internucleoside linking group(e.g., a phosphate group), or any derivative, analog, or modificationthereof that confers improved chemical and/or functional properties(e.g., binding affinity, nuclease resistance, chemical stability) to anucleic acid or a portion or segment thereof.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

As used herein, “parenteral administration,” “administeredparenterally,” and other grammatically equivalent phrases, refer tomodes of administration other than enteral and topical administration,usually by injection, and include, without limitation, intravenous,intranasal, intraocular, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural, intracerebral, intracranial,intracarotid and intrasternal injection and infusion.

As used herein, the term “patient” includes human and other mammaliansubjects that receive either prophylactic or therapeutic treatment.

The term “percent identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, the“percent identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared. For sequencecomparison, typically one sequence acts as a reference sequence to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra). In some embodiments, alignment of sequences isconducted by the Dawn method (Ricke, D. O. & Shcherbina, A. 2015 IEEEHigh Performance Extreme Computing Conference (HPEC),doi:10.1109.HPEC.2015.7322463 (2015)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

As generally used herein, “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

As used herein, a “pharmaceutically acceptable carrier” refers to, andincludes, any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Thecompositions can include a pharmaceutically acceptable salt, e.g., anacid addition salt or a base addition salt (see, e.g., Berge et al.(1977) J Pharm Sci 66:1-19).

As used herein, the terms “polypeptide,” “peptide”, and “protein” areused interchangeably to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

As used herein, the term “preventing” when used in relation to acondition, refers to administration of a composition which reduces thefrequency of, or delays the onset of, symptoms of a medical condition ina subject relative to a subject which does not receive the composition.

As used herein, the term “purified” or “isolated” as applied to any ofthe proteins described herein refers to a polypeptide that has beenseparated or purified from components (e.g., proteins or othernaturally-occurring biological or organic molecules) which naturallyaccompany it, e.g., other proteins, lipids, and nucleic acid in aprokaryote expressing the proteins. Typically, a polypeptide is purifiedwhen it constitutes at least 60 (e.g., at least 65, 70, 75, 80, 85, 90,92, 95, 97, or 99) %, by weight, of the total protein in a sample.

As used herein, the term “recombinant influenza vaccine” refers toinfluenza-specific immunogenic composition comprising one or more ofengineered influenza viral proteins described herein (e.g.,hemaglutinin, neuraminidase), including, but not limited to wholeinfluenza virus, subunit preparations thereof, virus-like particles,recombinant protein (i.e., preparations composed of recombinant HApurified to varying degree), and DNA- and viral vector-based vaccines.Recombinant influenza vaccines as described herein may optionallycontain one or more adjuvants.

As used herein, the term “subject” includes any human or non-humananimal. For example, the methods and compositions of the presentinvention can be used to treat a subject with an immune disorder. Theterm “non-human animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dog, cow, chickens,amphibians, reptiles, etc.

As used herein, the term “substantially” refers to the qualitativecondition of exhibiting total or near-total extent or degree of acharacteristic or property of interest. One of ordinary skill in thebiological arts will understand that biological and chemical phenomenararely, if ever, go to completion and/or proceed to completeness orachieve or avoid an absolute result. The term “substantially” istherefore used herein to capture the potential lack of completenessinherent in many biological and chemical phenomena.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available atwww.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50,60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percentidentity between two nucleotide or amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present disclosure canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See www.ncbi.nlm.nih.gov.

The term “T cell” refers to a type of white blood cell that can bedistinguished from other white blood cells by the presence of a T cellreceptor on the cell surface. There are several subsets of T cells,including, but not limited to, T helper cells (a.k.a. T_(H) cells orCD4⁺ T cells) and subtypes, including T_(H)1, T_(H)2, T_(H)3, T_(H)17,T_(H)9, and T_(FH) cells, cytotoxic T cells (i.e., Tc cells, CD8⁺ Tcells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memoryT cells and subtypes, including central memory T cells (T_(CM) cells),effector memory T cells (T_(EM) and T_(EMRA) cells), and resident memoryT cells (T_(RM) cells), regulatory T cells (a.k.a. T_(reg) cells orsuppressor T cells) and subtypes, including CD4⁺ FOXP3⁺ T_(reg) cells,CD4⁺ FOXP3⁻ T_(reg) cells, Tr1 cells, Th3 cells, and T_(reg)17 cells,natural killer T cells (a.k.a. NKT cells), mucosal associated invariantT cells (MAITs), and gamma delta T cells (γδ T cells), including Vγ9/Vδ2T cells. Any one or more of the aforementioned or unmentioned T cellsmay be the target cell type for a method of use of the invention.

As used herein, the terms “T cell activation” or “activation of T cells”refers to a cellular process in which mature T cells, which expressantigen-specific T cell receptors on their surfaces, recognize theircognate antigens and respond by entering the cell cycle, secretingcytokines or lytic enzymes, and initiating or becoming competent toperform cell-based effector functions. T cell activation requires atleast two signals to become fully activated. The first occurs afterengagement of the T cell antigen-specific receptor (TCR) by theantigen-major histocompatibility complex (MEW), and the second bysubsequent engagement of co-stimulatory molecules (e.g., CD28). Thesesignals are transmitted to the nucleus and result in clonal expansion ofT cells, upregulation of activation markers on the cell surface,differentiation into effector cells, induction of cytotoxicity orcytokine secretion, induction of apoptosis, or a combination thereof.

As used herein, the term “T cell-mediated response” refers to anyresponse mediated by T cells, including, but not limited to, effector Tcells (e.g., CD8⁺ cells) and helper T cells (e.g., CD4⁺ cells). T cellmediated responses include, for example, T cell cytotoxicity andproliferation.

As used herein, the terms “therapeutically effective amount” or“therapeutically effective dose,” or similar terms used herein areintended to mean an amount of an agent (e.g., a nucleic acid molecule)that will elicit the desired biological or medical response (e.g., animprovement in one or more symptoms of an infection).

The terms “treat,” “treating,” and “treatment,” as used herein, refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration to a subject, in need of suchtreatment, a human antibody of the present disclosure, for example, asubject in need of an enhanced immune response against a particularantigen or a subject who ultimately may acquire such a disorder, inorder to prevent, cure, delay, reduce the severity of, or ameliorate oneor more symptoms of the disorder or recurring disorder, or in order toprolong the survival of a subject beyond that expected in the absence ofsuch treatment.

As used herein, the term “vaccination” refers to the administration ofan immunogenic composition intended to generate an immune response, forexample to a disease-causing agent such as influenza. Vaccination can beadministered before, during, and/or after exposure to a disease-causingagent, and/or to the development of one or more symptoms, and in someembodiments, before, during, and/or shortly after exposure to the agent.Vaccines may elicit both prophylactic (preventative) and therapeuticresponses. Methods of administration vary according to the vaccine, butmay include inoculation, ingestion, inhalation or other forms ofadministration. Inoculations can be delivered by any of a number ofroutes, including parenteral, such as intravenous, subcutaneous orintramuscular. Vaccines may be administered with an adjuvant to boostthe immune response. In some embodiments, vaccination includes multipleadministrations, appropriately spaced in time, of an immunogeniccomposition.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Preferred methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the presently disclosed methods and compositions. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

EXAMPLES Example 1: Identification of Residues in H1N1

To identify highly conserved amino acid residues between strains of aparticular type and/or subtype of influenza virus, amino acid sequenceswere obtained and aligned. Specifically, the Dawn method, described inRicke, D. O & Shcherbina, A., IEEE High Performance Extreme ComputingConference (HPEC), doi:1031109/HPEC.2015.7322463 (2015), hereinincorporated by reference, was used to align 52,443 influenza A H1N1hemagglutinin amino acid sequences and 51,784 influenza A H1N1neuraminidase amino acid sequences. FIG. 1 shows an alignment of asection of amino acid residues in the H1N1 HA protein from strains inyears 2009-2019.

SEQ ID NO: 1 provides the amino acid sequence for hemagglutinin from theA/Michigan/45/2015 H1N1 strain. SEQ ID NO: 2 provides the amino acidsequence for neuraminidase from the A/Michigan/45/2015 strain H1N1strain.

Highly variable residues were identified for both proteins, along withresidues having low variability. The following sequence forhemagglutinin indicates hypervariable residues in bold and conservedregions are underlined.

(SEQ ID NO: 1) MKAILVVLLYTFTTANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGV APLHLGKCNIAGWI LGNPECESLSTASSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSF ERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLNQSYINDKGKEVLV LWGIHHPSTTADQQSLYQNADAYVFVGTSRYSKKFKPEIATRPKVRDQEGRMNYYWTLVEPGDK ITFEATGNLVVPRYAFTMERNAGSGIIISDTPVHDCNTTCQTPEGAINTSLPFQNIHPITIGKC PKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAAD LKS TQNAIDK ITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLEN ERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKL NREKIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI

FIG. 2 provides the amino acid sequence and nucleic acid sequences forthe H1N1 hemagglutinin protein with the nucleic acid sequencesunderlined for highly conserved regions and shown by boxes for thehypervariable amino acid residues.

The following sequence for neuraminidase indicates hypervariableresidues in bold and conserved regions are underlined.

(SEQ ID NO: 2) MNPNQKIITIGS ICMTIGMANLILQIGNIISIWVSHSIQIGNQSQIETCNQSVITYENNTWVNQ TYVNISNINFAAGQSVVSVKLAGNSSLCPVSGWAIYSKDNSV RIGSKGDVFVIREPFISCSPLE CR TFFLTQGALLNDKHSNGT I KDRSPYR TLMSCPIGEVPSPYNSR FESVAWSASACHDG IN WLT IGISGPD S GAVAVLKYNGIITDTIKSWRNN ILRTQESEC ACVNGSCFTIMIDGPSDGQASYKIF RIEKGKIIKSVEMKAPNYHYEECSCYPD SSEI TCVCRDNWHGSNRPWVSFNQNL EYQMGYICSG VTGDNPRPNDKTGSCGPVSSNGANGVKGFSFK YGNGVWIGRTKS ISSRK GFEMIWDPNGWT GTD NKFSIKQDIVGINE WSGYSGSFVQHPELTGL DCIRPCFWVEL IRGRPEENTIWTSGSSISFCGV NSDTVGWSWPDGAELPFTIDK

Example 2: Identification of Residues in H3N2

Using the same method described in Example 1, hypervariable amino acidresidues and highly conserved regions were identified in thehemagglutinin and neuraminidase proteins of H3N2. Specifically, 42,653hemagglutinin amino acid sequences and 29,491 neuraminidase amino acidsequences were aligned using the Dawn method.

SEQ ID NO: 3 provides the amino acid sequence for hemagglutinin from theA/Mississippi/27/2013 H3N2 strain. SEQ ID NO: 4 provides the amino acidsequence for neuraminidase from the Neuraminidase A/Miyagi/N1289/2005H3N2 strain.

Highly variable residues were identified for both proteins, along withresidues having low variability. The following sequence forhemagglutinin indicates hypervariable residues in bold and conservedregions are underlined.

(SEQ ID NO: 3) MKTIIALSYILCLVFAQKLPPYGNSTATLCLGHHALPNGTIVKTITNDRIEVTNATELVQNSSI GEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLV ASSGTLEFNNESFNWTGVTQNGTSSACIRRSNSSFFSRLNWLTHLNFKYPAINVIMPNNEQFDK LYIWGVHHPGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRNIPSRISIYWTIVKPG DILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGA CPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEG MVDGWYGFRHQNSEGRGQAADLK STQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALE NQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHNVYRDEAL NNRFQIKGVELKSGYKDWILWISFAISCFLLCVAIKGFIMWACQKGNIRCNIRCNICI

The following sequence for neuraminidase indicates hypervariableresidues in bold and conserved regions are underlined.

(SEQ ID NO: 4) MNPNQKIITIGSVSLTISTICFFMQIAILITTVTLHFKQYEFNSPPNNQVMLCEPTIIERNITE IVYLTNTTIEKEICPKLAEYRKWSKPQCNITGFAPFSKDNSIRLSAGGDIWVTREPYVSCDPDK CYQFALGQGTT LNNVHSNDIVRDRTPYRTLLMNELGVPFHLGTKQVCI AWSSSSCHDGKAWLHV CVTGDDKNATASFIYNGRLVDSIVSWSKEILRTQESECVCINGTCTVVMTDGSASGKADTKILF IEEGKIVHTSTLSGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIVDINIKDYSIVSSYVCSG LVGDTPRKNDSSSSSHCLDPNNEEGGHGVKGW AFDDGNDVWMGRTISEKLRSGYETFXVIEGWS NPNSKLQINRQVIVDRGNRSGYSGIFSVEGKSCINRCFYVELIRGRKEETEVLWTSNSIVVFCG TSGTYGTGSWPDGADINLMPI

Example 3: Identification of Residues in Influenza B

Using the same method described in Example 1, hypervariable amino acidresidues and highly conserved regions were identified in thehemagglutinin and neuraminidase proteins of influenza B. Specifically,20,906 hemagglutinin amino acid sequences and 14,546 neuraminidase aminoacid sequences were aligned using the Dawn method.

SEQ ID NO: 5 provides the amino acid sequence for hemagglutinin from theB/Brisbane/60/2008 influenza B strain. SEQ ID NO: 6 provides the aminoacid sequence for neuraminidase from the B/Wisconsin/05/2016 influenza Bstrain.

Highly variable residues were identified for both proteins, along withresidues having low variability. The following sequence forhemagglutinin indicates hypervariable residues in bold and conservedregions are underlined.

(SEQ ID NO: 5) MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTET RGKLCPKCL NCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQL P NLLRG YE HIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFATMAWAVP KNDKNKTATNPLTIEV PYICTEGEDQITVWGFHSDDETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLP QSGRIVVDYM VQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHE K YGGLNK SKPYYTG EHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWH GYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDD LRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVE IGNGCFETKHKCNQTCLD RIAAGTFD AGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLM IAIFVVYMVSR DNVSCSICL

The following sequence for neuraminidase indicates hypervariableresidues in bold and conserved regions are underlined.

(SEQ ID NO: 6) MLPSTIQTLTLFLTSGGVLLSLYVSASLSYLLYSDILLKFSPTEITAPTMPLDCANASNVQAVN RSATKGVTLLLLPEPEWTYPRLSCPGSTFQKALLISPHRFGE TKGNSAPLIIREPFVACGPNEC KHFALTHYAAQPGGYYNGTRG DRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDG KEWTYI GVDGPDNNALLKV KYGEAYTDTYHSYANNILRTQESACNCIGGNCYLMITDGSASGVSECRFLK IREGRIIKEIFPTGRVKHTEECTCGFASNKTIECACRDNR YTAKRPFVKLNVETDTAEIRLMCT DTYLDTPRPNDGSITGPCESDGDKGSGGIKGGFVHQRMKSKIGRWYSRTMSKTERMGMGLYVKY GGDPWADSDALAFSGVMVSMK EPGWYSFGFEIKDKKCDVPCIGIEMVHDGGKETWHSAATAIYC LMGSGQLLWDTVTGVDMAL

Example 4: Production of B Cell Immune Response

To determine whether the hypervariable residues identified in HA and NAproteins as described in Examples 1-3, alanine scanning of each residueand combinations of residues is performed. FIG. 3 shows an exemplarysequence wherein each hypervariable residue identified in the H1N1 HAprotein described in Example 1 is replaced with an alanine.

Each mutated HA and NA protein comprising an alanine is subjected to invitro and in vivo testing to determine what mutations will elicit animmune response to highly conserved amino acid regions and provideprotection against influenza infection.

In one study, mutated HA or NA proteins, or combinations thereof, areadministered to a subject (e.g., a pig). Serum, BAL, and TBLN samplesare collected and tested in ELISA or neutralization assays to determineantibodies titers to the highly conserved amino acid regions. Generationof such antibodies indicates the immune response has been directed tosuch regions and thus the mutated proteins are suitable as a universalinfluenza vaccine. In another study, after administration of the mutatedHA or NA proteins, or combinations thereof, subjects are challenged withvarious influenza virus strains and infection levels are monitored. Theability of the mutated HA or NA proteins to prevent infection bydifferent influenza strains indicates the mutated proteins are suitableas a universal influenza vaccine.

Example 5: Production of T Cell Immune Response

To determine whether the highly conserved regions of amino acidsidentified in Examples 1-3 are capable of eliciting a T cell immuneresponse, immunogenic compositions comprising polypeptides having aminoacid sequences of the conserved regions are generated and administeredto subjects. In some studies, polypeptides comprising different regionsare combined by operably linking the polypeptides together.

PBMCS are collected at various time points after immunization, and arecultured with 15-mer peptide pools encompassing the sequence of thepolypeptide or operably linked polypeptides. T cell activation ismeasured by assessing proliferation, production of cytokines and/or thecytotoxic ability of the cells against different influenza virusstrains. The ability of the polypeptide or operably linked polypeptidesto induce cytokine induction or induce killing of different strains by Tcells indicates the polypeptide(s) are suitable as a universal influenzavaccine.

In another study, polypeptide(s) or operably linked polypeptides areadministered to a subject (e.g., a pig) which is then challenged withvarious influenza virus strains and infection levels are monitored. Theability of the polypeptide(s) to prevent infection by differentinfluenza strains indicates they are suitable as a universal influenzavaccine.

Example 6: Therapeutic Efficacy of Nucleic Acids Targeting HighlyConserved Regions

To determine whether targeting the highly conserved regions identifiedin Examples 1-3 provides therapeutic efficacy, nucleic acid molecules(e.g., siRNA or miRNA) having substantial complementarity to nucleotidesequences encoding the highly conserved regions are generated.

In one study, a nucleic acid molecule targeting a highly conservedregion is contacted with various influenza virus strains. Ability of theviruses to infect cells is assessed after contact. If the nucleic acidmolecule disrupts the life cycle of the virus and prevents infection,the nucleic acid molecule may be suitable for treating influenzainfection.

SEQUENCE LISTING TABLE SEQ ID NO Description Sequence 1 H1N1MKAILVVLLYTFTTANADTLCIGYHANNSTDT HemagglutininVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGV A/Michigan/ APLHLGKCNIAGWILGNPECESLSTASSWSYI 45/2015 strain VETSNSDNGTCYPGDFINYEELREQLSSVSSF(amino acid) ERFEIFPKTSSWPNHDSNKGVTAACPHAGAKS GenBank:FYKNLIWLVKKGNSYPKLNQSYINDKGKEVLV MK622940.1LWGIHHPSTTADQQSLYQNADAYVFVGTSRYS KKFKPEIATRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFTMERNAGSGIIISDTP VHDCNTTCQTPEGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAG FIEGGWTGMVDGWYGYHHQNEQGSGYAAD LKSTQNAIDK ITNKVNSVIEKMNTQFTAVGKEFNH LEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNG CFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVKLESTRIYQILAIYSTVASSLVLV VSLGAISFWMCSNGSLQCRICI(underline = highly conserved; bold = hypervariable) 2 H1N1 MNPNQKIITIGSICMTIGMANLILQIGNIISI Neuraminidase WVSHSIQIGNQSQIETCNQSVITYENNTWVNQA/Michigan/ TYVNISNINFAAGQSVVSVKLAGNSSLCPVSG 45/2015 strain WAIYSKDNSVRIGSKGDVFVIREPFISCSPLE (amino acid) CR TFFLTQGALLNDKHSNGT I KDRSPYR TLMSGenBank: CPIGEVPSPYNSR FESVAWSASACHDG IN WLT MK622934.1 IGISGPD SGAVAVLKYNGIITDTIKSWRNN IL RTQESEC ACVNGSCFTIMIDGPSDGQASYKIFRIEKGKIIKSVEMKAPNYHYEECSCYPD SSEI T CVCRDNWHGSNRPWVSFNQNL EYQMGYICSGVTGDNPRPNDKTGSCGPVSSNGANGVKGFSFK YG NGVWIGRTKS ISSRK GFEMIWDPNGWT GTDNKFSIKQDIVGINE WSGYSGSFVQHPELTGL D CIRPCFWVEL IRGRPEENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK (underline = highly conserved;bold = hypervariable) 3 H3N2 MKTIIALSYILCLVFAQKLPPYGNSTATLCLGHemagglutinin HHALPNGTIVKTITNDRIEVTNATELVQNSSI A/Mississippi/GEICDSPHQILDGENCTLIDALLGDPQCDGFQ 27/2013 strainNKKWDLFVERSKAYSNCYPYDVPDYASLRSLV (amino acid)ASSGTLEFNNESFNWTGVTQNGTSSACIRRSN GenBank:SSFFSRLNWLTHLNFKYPAINVIMPNNEQFDK AIK26600.1LYIWGVHHPGTDKDQIFLYAQSSGRITVSTKR SQQAVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDA PIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIA GFIENGWEG MVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFS EVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGN GCFKIYHKCDNACIGSIRNGTYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLC VAIKGFIMWACQKGNIRCNIRCNICI(underline = highly conserved; bold = hypervariable) 4 H3N2MNPNQKIITIGSVSLTISTICFFMQIAILITT NeuraminidaseVTLHFKQYEFNSPPNNQVMLCEPTIIERNITE A/Miyagi/N12IVYLTNTTIEKEICPKLAEYRKWSKPQCNITG 89/2005 strainFAPFSKDNSIRLSAGGDIWVTREPYVSCDPDK (amino acid) CYQFALGQGTTLNNVHSNDIVRDRTPYRTLLM GenBank: NELGVPFHLGTKQVCI AWSSSSCHDGKAWLHVAB271522.1 CVTGDDKNATASFIYNGRLVDSIVSWSKEILRTQESECVCINGTCTVVMTDGSASGKADTKILF IEEGKIVHTSTLSGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIVDINIKDYSIVSSYVCSG LVGDTPRKNDSSSSSHCLDPNNEEGGHGVKGW AFDDGNDVWMGRTISEKLRSGYETFXVIEGWS NPNSKLQINRQVIVDRGNRSGYSGIFSVEGKSCINRCFYVELIRGRKEETEVLWTSNSIVVFCG TSGTYGTGSWPDGADINLMPI(underline = highly conserved; bold = hypervariable) 5 Influenza BMKAIIVLLMVVTSNADRICTGITSSNSPHVVK HemagglutininTATQGEVNVTGVIPLTTTPTKSHFANLKGTET B/Brisbane/60/ RGKLCPKCLNCTDLDVALGRPKCTGKIPSARV 2008 strain SILHEVRPVTSGCFPIMHDRTKIRQL P NLLRG(amino acid) YE HIRLSTHNVINAENAPGGPYKIGTSGSCPN GenBank: ITNGNGFFATMAWAVPKNDKNKTATNPLTIEV KX058884.1 PYICTEGEDQITVWGFHSDDETQMAKLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLP QSGRIVVDYM VQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLIGEADCLHE K YGGLNK SKPYYTG EHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWH GYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDD LRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVE IGNGCFETKHKCNQTCLD RIAAGTFD AGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLM IAIFVVYMVSR DNVSCSICL(underline = highly conserved; bold = hypervariable) 6 Influenza BMLPSTIQTLTLFLTSGGVLLSLYVSASLSYLL NeuraminidaseYSDILLKFSPTEITAPTMPLDCANASNVQAVN B/Wisconsin/RSATKGVTLLLLPEPEWTYPRLSCPGSTFQKA 05/2016 strain LLISPHRFGETKGNSAPLIIREPFVACGPNEC (amino acid) KHFALTHYAAQPGGYYNGTRG DRNKLRHLISVGenBank: KLGKIPTVENSIFHMAAWSGSACHDG KEWTYI KX007164.1 GVDGPDNNALLKVKYGEAYTDTYHSYANNILR TQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRIIKEIFPTGRVKHTEECTCGFASNKTI ECACRDNR YTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPCESDGDKGSGGIKGG FVHQRMKSKIGRWYSRTMSKTERMGMGLYVKYGGDPWADSDALAFSGVMVSMK EPGWYSFGFEI KDKKCDVPCIGIEMVHDGGKETWHSAATAIYCLMGSGQLLWDTVTGVDMAL (underline = highly conserved; bold = hypervariable)7 H1N1 GYHANNST Hemagglutinin conserved region (amino acid) 8 H1N1ggttatcatgcgaacaattcaaca Hemagglutinin conserved region (nucleic acid) 9H1N1 NVTVTHS Hemagglutinin conserved region (amino acid) 10 H1N1aatgtaacagtaacacactct Hemagglutinin conserved region (nucleic acid) 11H1N1 SWSYIVE Hemagglutinin conserved region (amino acid) 12 H1N1tcatggtcctacattgtggaa Hemagglutinin conserved region (nucleic acid) 13H1N1 QSRGLFGAIAGF Hemagglutinin conserved region (amino acid) 14 H1N1caatctagaggcctattcggggccattgccggcttc Hemagglutinin conserved region(nucleic acid) 15 H1N1 QGSGYAAD Hemagglutinin conserved region (aminoacid) 16 H1N1 caggggtcaggatatgcagccgac Hemagglutinin conserved region(nucleic acid) 17 H1N1 ITNKVNS Hemagglutinin conserved region (aminoacid) 18 H1N1 attactaacaaagtaaattct Hemagglutinin conservedregion (nucleic acid) 19 H1N1 WTYNAELL Hemagglutinin conserved region(amino acid) 20 H1N1 tggacttacaatgccgaactgttg Hemagglutinin conservedregion (nucleic acid) 21 H1N1 GCFEFYH Hemagglutinin conserved region(amino acid) 22 H1N1 gcctgctttgaattttaccac Hemagglutinin conservedregion (nucleic acid) 23 H1N1 LGNPEC Hemagglutinin conserved region(amino acid) 24 H1N1 ctgggaaatccagagtgt Hemagglutinin conserved region(nucleic acid) 25 H1N1 EGGWTG Hemagglutinin conserved region (aminoacid) 26 H INI gaaggggggtggacaggg Hemagglutinin conserved region(nucleic acid) 27 H1N1 LLENER Hemagglutinin conserved region (aminoacid) 28 H1N1 ctattggaaaatgaaaga Hemagglutinin conserved region (nucleicacid) 29 H1N1 MNPNQKI1TIGS Neuraminidase conserved region (amino acid)30 H1N1 atgaatccaaaccaaaagataataaccattggttcg Neuraminidase conservedregion (nucleic acid) 31 H1N1 RIGSKGDVFV Neuraminidase conservedregion (amino acid) 32 H1N1 agaatcggttccaagggggatgtgtttgtc Neuraminidaseconserved region (nucleic acid) 33 H1N1 REPFISCS Neuraminidase conservedregion (amino acid) 34 H1N1 agggaaccattcatatca Neuraminidase conservedregion (nucleic acid) 35 H1N1 TFFLTQGALLNDKHSNGT Neuraminidase conservedregion (amino acid) 36 H1N1 accttcttcttgactcaaggggccttgctaaaNeuraminidase tgacaaacattccaatggaacc conserved region (nucleic acid) 37H1N1 KDRSPYR Neuraminidase conserved region (amino acid) 38 H1N1aaagacaggagcccataccga Neuraminidase conserved region (nucleic acid) 39H1N1 FESVAWSASACHDG Neuraminidase conserved region (amino acid) 40 H1N1tttgagtcagtcgcttggtcagcaagtgcttgtcatgatggc Neuraminidase conservedregion (nucleic acid) 41 H1N1 WLTIGISGPD Neuraminidase conserved region(amino acid) 42 H1N1 tggctaacaattggaatttctggcccagac Neuraminidaseconserved region (nucleic acid) 43 H1N1 ILRTQESEC Neuraminidaseconserved region (amino acid) 44 H1N1 atattgagaacacaagagtctgaatgtNeuraminidase conserved region (nucleic acid) 45 H1N1 YEECSCYPDNeuraminidase conserved region (amino acid) 46 H1N1tatgaggaatgctcctgttaccctgat Neuraminidase conserved region (nucleicacid) 47 H1N1 CVCRDNWHGSNRPWVSFNQNL Neuraminidase conserved region(amino acid) 48 H1N1 tgtgtgtgcagggataactggcatggctcga Neuraminidaseatcgaccgtgggtgtctttcaaccagaatctg conserved region (nucleic acid) 49 H1N1NGVWIGRTKS Neuraminidase conserved region (amino acid) 50 H1N1aatggtgtttggatagggagaactaaaagc Neuraminidase conserved region (nucleicacid) 51 H1N1 GFEMIWDPNGWT Neuraminidase conserved region (amino acid)52 H1N1 ggttttgagatgatttgggatccgaatggatggact Neuraminidase conservedregion (nucleic acid) 53 H1N1 WSGYSGSFVQHPELTGL Neuraminidase conservedregion (amino acid) 54 H1N1 tggtcagggtatagcgggagttttgttcagcatccNeuraminidase agaactaacagggctg conserved region (nucleic acid) 55 H1N1RPCFWVEL Neuraminidase conserved region (amino acid) 56 H1N1agaccttgcttctgggttgaacta Neuraminidase conserved region (nucleic acid)57 H1N1 WTSGSS1SFCGV Neuraminidase conserved region (amino acid) 58 H1N1tggactagcgggagcagcatatccttttgtggtgta Neuraminidase conserved region(nucleic acid) 59 H1N1 WSWPDGAELPF Neuraminidase conserved region (aminoacid) 60 H1N1 tggtcttggccagacggtgctgagttgccattt Neuraminidase conservedregion (nucleic acid) 61 H3N2 LCLGHHA Hemagglutinin conserved region(amino acid) 62 H3N2 ctgtgccttgggcaccatgcatta Hemagglutinin conservedregion (nucleic acid) 63 H3N2 GNLIAPRGYF Hemagglutinin conserved region(amino acid) 64 H3N2 gggaatctaattgctcctaggggttacttc Hemagglutininconserved region (nucleic acid) 65 H3N2 LKLATGMRN Hemagglutininconserved region (amino acid) 66 H3N2 ctgaaattggcaacaggaatgcgaaatHemagglutinin conserved region (nucleic acid) 67 H3N2 FGAIAGFIENGWEGHemagglutinin conserved region (amino acid) 68 H3N2tttggcgcaatagcaggtttcatagaaaatggttgggagggg Hemagglutinin conservedregion (nucleic acid) 69 H3N2 KFHQIEKEF Hemagglutinin conserved region(amino acid) 70 H3N2 aaattccatcagattgaaaaagaattc Hemagglutinin conservedregion (nucleic acid) 71 H3N2 DLTDSEM Hemagglutinin conserved region(amino acid) 72 H3N2 gatctaactgactcagaaatg Hemagglutinin conservedregion (nucleic acid) 73 H3N2 LRENAED Hemagglutinin conserved region(amino acid) 74 H3N2 ctgagggaaaatgctgaggat Hemagglutinin conservedregion (nucleic acid) 75 H3N2 QFALGQGTT Neuraminidase conserved region(amino acid) 76 H3N2 caatttgcccttggacagggaacaaca Neuraminidase conservedregion (nucleic acid) 77 H3N2 AWSSSSC Neuraminidase conserved region(amino acid) 78 H3N2 gcatggtccagctcaagttgt Neuraminidase conservedregion (nucleic acid) 79 H3N2 LRTQESEC Neuraminidase conserved region(amino acid) 80 H3N2 ctcaggacccaggagtcagaatgc Neuraminidase conservedregion (nucleic acid) 81 H3N2 EECSCYP Neuraminidase conserved region(amino acid) 82 H3N2 gaggagtgctcctgctatcct Neuraminidase conservedregion (nucleic acid) 83 H3N2 CSGLVGDTPR Neuraminidase conserved region(amino acid) 84 H3N2 tgctcaggacttgttggagacacacccaga Neuraminidaseconserved region (nucleic acid) 85 H3N2 GVKGWAFD Neuraminidase conservedregion (amino acid) 86 H3N2 ggagtgaaaggctgggcctttgat Neuraminidaseconserved region (nucleic acid) 87 H3N2 NRCFYVELIRG Neuraminidaseconserved region (amino acid) 88 H3N2 aatcggtgcttttatgtggagttgataaggggaNeuraminidase conserved region (nucleic acid) 89 H3N2 VFCGTSGTYGNeuraminidase conserved region (amino acid) 90 H3N2gtgttttgtggcacctcaggtacatatgga Neuraminidase conserved region (nucleicacid) 91 H3N2 GSWPDGA Neuraminidase conserved region (amino acid) 92H3N2 ggctcatggcctgatggggcg Neuraminidase conserved region (nucleic acid)93 Influenza B VKTATQEVNVTG Hemagglutinin conserved region (amino acid)94 Influenza B gtcaaaactgctactcaaggggaggtcaatgtgactggt Hemagglutininconserved region (nucleic acid) 95 Influenza B NCTDLDVAL Hemagglutininconserved region (amino acid) 96 Influenza B aactgcacagatctggacgtagccttgHemagglutinin conserved region (nucleic acid) 97 Influenza BTSGCFPIMHDRTKIRQL Hemagglutinin conserved region (amino acid) 98Influenza B acatctgggtgctttcctataatgcacgac Hemagglutininagaacaaaaattagacagctg conserved region (nucleic acid) 99 Influenza BNLLRGYE Hemagglutinin conserved region (amino acid) 100 Influenza Baaccttctccgaggatacgaa Hemagglutinin conserved region (nucleic acid) 101Influenza B TMAWAVP Hemagglutinin conserved region (amino acid) 102Influenza B acaatggcttgggccgtccca Hemagglutinin conserved region(nucleic acid) 103 Influenza B EDGGLPQSGRIVVDYM Hemagglutinin conservedregion (amino acid) 104 Influenza B gaagacggaggactaccacaaagtggtaHemagglutinin gaattgttgttgattacatg conserved region (nucleic acid) 105Influenza B LPLIGEADCLHE Hemagglutinin conserved region (amino acid) 106Influenza B ttgcctttaattggagaagcagattgcctccacgaa Hemagglutinin conservedregion (nucleic acid) 107 Influenza B YGGLNKSKPYYTG Hemagglutininconserved region (amino acid) 108 Influenza Btacggtggattaaacaaaagcaagccttactacacaggg Hemagglutinin conserved region(nucleic acid) 109 Influenza B CPIWVKTPL Hemagglutinin conserved region(amino acid) 110 Influenza B tgcccaatatgggtgaaaacacccttg Hemagglutininconserved region (nucleic acid) 111 Influenza B GFFGAIAGFLEGGWEGMHemagglutinin conserved region (amino acid) 112 Influenza Bggtttcttcggagctattgctggtttcttag Hemagglutinin aaggaggatgggaaggaatgconserved region (nucleic acid) 113 Influenza B AGWHGYTSHGAHGHemagglutinin conserved region (amino acid) 114 Influenza Bgcaggttggcacggatacacatcccatggggcacatgga Hemagglutinin conserved region(nucleic acid) 115 Influenza B AVAADLKSTQEA Hemagglutinin conservedregion (amino acid) 116 Influenza B gcggtggcagcagaccttaagagcactcaagaggccHemagglutinin conserved region (nucleic acid) 117 Influenza BKITKNLNSLSELE Hemagglutinin conserved region (amino acid) 118Influenza B aagataacaaaaaatctcaactctttgagtgagctggaa Hemagglutininconserved region (nucleic acid) 119 Influenza B KNLQRLS Hemagglutininconserved region (amino acid) 120 Influenza B aagaatcttcaaagactaagcHemagglutinin conserved region (nucleic acid) 121 Influenza BEILELDEKVDDLRADTISSQIELAVLLSNEGIINSED Hemagglutinin EHLLALERKLKKMLGPSAconserved region (amino acid) 122 Influenza Bgaaatactagaactagatgagaaagtggatga Hemagglutinintctcagagctgatacaataagctcacaaatag conservedaactcgcagtcctgctttccaatgaaggaata region ataaacagtgaagatgaacatctcttggcgct(nucleic tgaaagaaagctgaagaaaatgctgggcccct acid) ctgct 123 Influenza BIGNGCFETKHKCNQTCLD Hemagglutinin conserved region (amino acid) 124Influenza B atagggaatggatgctttgaaaccaaac Hemagglutininacaagtgcaaccagacctgtctcgac conserved region (nucleic acid) 125Influenza B AGEFSLPTFDSLNITAASL Hemagglutinin conserved region (aminoacid) 126 Influenza B gcaggagaattttctctccccacctttg Hemagglutininattcactgaatattactgctgcatcttta conserved region (nucleic acid) 127Influenza B HTILLYYSTAASSLAVTLM Hemagglutinin conserved region (aminoacid) 128 Influenza B catactatactgctttactactcaactgc Hemagglutinintgcctccagtttggctgtaacactgatg conserved region (nucleic acid) 129Influenza B ALLISPHRFGE Neuraminidase conserved region (amino acid) 130Influenza B gcactcctaattagccctcatagattcggagaa Neuraminidase conservedregion (nucleic acid) 131 Influenza B HFALTHYAAQPG Neuraminidaseconserved region (amino acid) 132 Influenza Bcactttgctttaacccattatgcagcccaaccaggg Neuraminidase conserved region(nucleic acid) 133 Influenza B DRNKLRHL Neuraminidase conserved region(amino acid) 134 Influenza B gacagaaacaagctgaggcatcta Neuraminidaseconserved region (nucleic acid) 135 Influenza B AWSGSACHDG Neuraminidaseconserved region (amino acid) 136 Influenza Bgcatggagcgggtccgcgtgccatgatggt Neuraminidase conserved region (nucleicacid) 137 Influenza B KYGEAYTDTYHSY Neuraminidase conserved region(amino acid) 138 Influenza B aaatatggagaagcatatactgacacataccattcctatNeuraminidase conserved region (nucleic acid) 139 Influenza BLRTQESACNCI Neuraminidase conserved region (amino acid) 140 Influenza Bctaagaacacaagaaagtgcctgcaattgcatc Neuraminidase conserved region(nucleic acid) 141 Influenza B CRFLKIREGR Neuraminidase conserved region(amino acid) 142 Influenza B tgcagatttcttaagattcgagagggccgaNeuraminidase conserved region (nucleic acid) 143 Influenza B HTEECTCGFANeuraminidase conserved region (amino acid) 144 Influenza Bcacactgaggaatgcacatgcggatttgcc Neuraminidase conserved region (nucleicacid) 145 Influenza B YTAKRPFVKL Neuraminidase conserved region (aminoacid) 146 Influenza B tacacagcaaaaagaccttttgtcaaatta Neuraminidaseconserved region (nucleic acid) 147 Influenza B KGGFVHQR Neuraminidaseconserved region (amino acid) 148 Influenza B aagggaggatttgttcatcaaagaNeuraminidase conserved region (nucleic acid) 149 Influenza B GRWYSRTNeuraminidase conserved region (amino acid) 150 Influenza Bggaaggtggtactctcgaacg Neuraminidase conserved region (nucleic acid) 151Influenza B EPGWYSFGFE Neuraminidase conserved region (amino acid) 152Influenza B gaacctggttggtattcctttggcttcgaa Neuraminidase conservedregion (nucleic acid) 153 Influenza B EMVHDGG Neuraminidase conservedregion (amino acid) 154 Influenza B gagatggtacatgatggtgga Neuraminidaseconserved region (nucleic acid) 155 H1N1 GAVAVLKY Neuraminidaseconserved region (amino acid) 156 H1N1 ggggcagtggctgtgttaaagtacNeuraminidase conserved region (nucleic acid) 157 Variant ofMKAILVVLLYTFAAANADTLCIGYH H1N1 ANNSTDTVDTVLEKNVTVTHSVNLL HemagglutininEAAHNGKLCKLRGVAPLHLGKCNIA A/Michigan/45/ GWALGNPECEALATASSWSYIVETS2015 strain ASDNGTCYPGDFIAYEELREQLSSV (amino acid)SSFERFEIFPKASSWPNHDANAGVT GenBank: AACPAAGAAAFYANLIWLVKKGNSY MK622940.1PKAAASYINAKAKEVLVLWAIHHPA Hypervariable TAADQQSLYQNADAYVFVGASAYSAresidues KFAPEIAARPKVRAQAGRMNYYWTL substituted AEPGDAITFEATGNLVVPRYAFAAAwith Ala RAAGSGIIISDAAVHDCATTCQTPA GAINTSLPFQNIHPATIGACPKYVKSTKLRAATGLRNAPSIQSRGLFGAI AGFIEGGWTGMADGWYGYHHQNEQGSGYAADAKSTQNAIDAITNKVNSVI EKMNTQFTAVGKEFAHLEARIENLNKKVDDGFLDIWTYNAELLVLLENER TLDYHDSNVKNLYEKVRAQLKNNAKEIGNGCFEFYHKCDAACMESVKNGT YDYPKYSEEAKLNREAIDGVKLESTRIYQILAIYSTVASSLVLAVSLGAI SFWMCSNGSLQCRICI* 158 Influenza ASNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYIN H1N1DKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYS Hemagglutinin KKFKP2009 residues 145-229 159 Influenza ASNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYIN H1N1DKGKEVLVLWGIHHPPTSADQQSLYQNADAYVFVGTSRYS Hemagglutinin KKFKP2010 residues 145-229 160 Influenza ATTRGTTVACSHSGANSFYRNLLWIVKKGNSYPKLSKSYTNN H1N1KGKEVLVIWGVHHPPTDSDQQTLYQNNHTYVSVGSSKYYK Hemagglutinin RLTP2011 residues 145-229 161 Influenza ASNKGVTAACPHAGAKGFYKNLIWLVKKGNSYPKLSKSYIN H1N1DKGKEVLVLWGIHHPSTTADQQSLYQNADTYWVGTSRYS Hemagglutinin KKFKP2012 residues 145-229 162 Influenza ASNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYIN H1N1DKGKEVLVLWGIHHPSTTADQQSLYQNANAYVFVGTSKYS Hemagglutinin KKFKP2013 residues 145-229 163 Influenza ASNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYIN H1N1DKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGTSRYS Hemagglutinin KKFKP2014 residues 145-229 164 Influenza ASNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYIN H1N1DKGKEVLVLWGIHHPSTSADQQSLYQNADAYWVGTSRYS Hemagglutinin KKFKP2015 residues 145-229 165 Influenza ASNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLNQSYIN H1N1DKGKEVLVLWGIHHPSTTADQQSLYQNADAYVFVGTSRYS Hemagglutinin KKFKP2016 residues 145-229 166 Influenza ASNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLNQTYIN H1N1DKGKEVLVLWGIHHPPTTADQQSLYQNADAYVFVGTSRYS Hemagglutinin KKFKP2017 residues 145-229 167 Influenza ASDKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLNQTYIN HIN1DKGKEVLVLWGIHHPPTIADQQSLYQNADAYVFVGTSRYS Hemagglutinin KKFKP2018 residues 145-229 168 Influenza ASNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKINQTYIND H1N1KGKEVLVLWGIHHPPTTADOQSLYQNADAYVFVGTSRYSK Hemagglutinin KFKP2019 residues 145-229 169 H1N1 atgaaggcaatactagtagttctgctatatHemagglutinin acatttacaaccgcaaatgcagacacatta A/Michigan/tgtataggttatcatgcgaacaattcaaca 45/2015 straingacactgtagacacagtactagaaaagaat (nucleotide)gtaacagtaacacactctgttaaccttctg GenBank: gaagacaagcataacggaaaactatgcaaaMK622940.1 ctaagaggggtagccccattgcatttgggt aaatgtaacattgctggctggatcctgggaaatccagagtgtgaatcrctctccacagca agttcatggtcctacattgtggaaacatctaattcagacaatggaacgtgttacccagga gatttcatcaattatgaggagctaagagagcaattgagctcagtgtcatcatttgaaagg tttgagatattccccaagacaagttcatggcccaatcatgactcgaacaaaggtgtaacg gcagcatgtcctcacgctggagcaaaaagcttctacaaaaacttgatatggctagttaaa aaaggaaattcatacccaaagcttaaccaatcctacattaatgataaagggaaagaagtc ctcgtgctgtggggcattcaccatccatctactactgctgaccaacaaagtctctatcag aatgcagatgcatatgtttttgtggggacatcaagatacagcaagaagttcaagccggaa atagcaacaagacccaaagtgagggatcaagaagggagaatgaactattactggacacta gtagagccgggagacaaaataacattcgaagcaactggaaatctagtggtaccgagatat gcattcacaatggaaagaaatgctggatctggtattatcatttcagatacaccagtccac gattgcaatacaacttgtcagacacccgagggtgctataaacaccagcctcccatttcag aatatacatccgatcacaattggaaaatgtccaaagtatgtaaaaagcacaaaattgaga ctggccacaggattgaggaatgttccgtctattcaatctagaggcctattcggggccatt gccggcttcattgaaggggggtggacagggatggtagatggatggtacggttatcaccat caaaatgagcaggggtcaggatatgcagccgacctgaagagcacacaaaatgccattgac aagattactaacaaagtaaattctgttattgaaaagatgaatacacagttcacagcagtg ggtaaagagttcaaccacctggaaaaaagaatagagaatctaaataaaaaagttgatgat ggtttcctggacatttggacttacaatgccgaactgttggttctattggaaaatgaaaga actttggactatcacgattcaaatgtgaagaacttgtatgaaaaagtaagaaaccagtta aaaaacaatgccaaggaaattggaaacggctgctttgaattttaccacaaatgcgataac acgtgcatggaaagtgtcaaaaatgggacttatgactacccaaaatactcagaggaagca aaattaaacagagaaaaaatagatggggtaaagctggaatcaacaaggatttaccagatt ttggcgatctattcaactgtcgccagttcattggtactggtagtctccctgggggcaatc agcttctggatgtgctctaatgggtctctacagtgtagaatatgtatttaa 170 Variant of atgaaggcaatactagtagttctgctatat H1N1acatttgcagccgcaaatgcagacacatta Hemagglutinintgtataggttatcatgcgaacaattcaaca A/Michigan/gacactgtagacacagtactagaaaagaat 45/2015 gtaacagtaacacactctgttaaccttctgstrain gaagccgcgcataacggaaaactatgcaaa (nucleotide)ctaagaggggtagccccattgcatttgggt GenBank: aaatgtaacattgctggctgggccctgggaMK622940.1 aatccagagtgtgaagcrctcgccacagca Hypervariableagttcatggtcctacattgtggaaacatct residues gcttcagacaatggaacgtgttacccaggasubstituted gatttcatcgcttatgaggagctaagagag with Alacaattgagctcagtgtcatcatttgaaagg tttgagatattccccaaggcaagttcatggcccaatcatgacgcgaacgcaggtgtaacg gcagcatgtcctgccgctggagcagcagccttctacgcaaacttgatatggctagttaaa aaaggaaattcatacccaaaggctgccgcatcctacattaatgctaaagcgaaagaagtc ctcgtgctgtgggccattcaccatccagctactgctgctgaccaacaaagtctctatcag aatgcagatgcatatgtttttgtggggacatcagcatacagcgcgaagttcgcgccggaa atagcagcaagacccaaagtgagggctcaagcagggagaatgaactattactggacacta gcagagccgggagacgcaataacattcgaagcaactggaaatctagtggtaccgagatat gcattcgcagcggcaagagctgctggatctggtattatcatttcagatgcagcagtccac gattgcgctacaacttgtcagacacccgcgggtgctataaacaccagcctcccatttcag aatatacatccggccacaattggagcatgtccaaagtatgtaaaaagcacaaaattgaga gcggccacaggattgaggaatgctccgtctattcaatctagaggcctattcggggccatt gccggcttcattgaaggggggtggacagggatggcagatggatggtacggttatcaccat caaaatgagcaggggtcaggatatgcagccgacgcgaagagcacacaaaatgccattgac gcgattactaacaaagtaaattctgttattgaaaagatgaatacacagttcacagcagtg ggtaaagagttcgcccacctggaagcaagaatagagaatctaaataaaaaagttgatgat ggtttcctggacatttggacttacaatgccgaactgttggttctattggaaaatgaaaga actttggactatcacgattcaaatgtgaagaacttgtatgaaaaagtaagagcccagtta aaaaacaatgccaaggaaattggaaacggctgctttgaattttaccacaaatgcgatgcc gcgtgcatggaaagtgtcaaaaatgggacttatgactacccaaaatactcagaggaagca aaattaaacagagaagcaatagatggggtaaagctggaatcaacaaggatttaccagatt ttggcgatctattcaactgtcgccagttcattggtactggcagtctccctgggggcaatc agcttctggatgtgctctaatgggtctctacagtgtagaatatgtatttaa 171 H3N2 M1 MSLLTEVETYVLSI V PSGPLKAEIAQRLE DVFAGKNTDLEAL Protein MEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMD KAVKLYR KLKREITFHGAKE IALSYSA GAL ASCMGLIYNRMG AVTTEVAFGLVCATCEQIADSQHRSHRQ M VATTNPLIKHENRMVLASTTAKAMEQMAGSSEQAAEAM EIASQARQMVQAMRAI GTHPSSS TGLR DDLLENLQ T YQKR MGVQMQRFK(underline = highly conserved; bold = hypervariable) 172 H3N2 NEPMDSNTVSSFQDIL L RMSKMQLGSSSEDLNGMITQFESLKI Y Protein RDSLGE AVMRMGDLHLLQNRNGKWREQLGQKFEEIRWLIE EVRHRLRTTENSFEQITFMQALQLL F EVEQEIRTFSFQLI(underline = highly conserved; bold = hypervariable) 173 H3N2 NPMASQGTKRSYEQMET DGDRQNATEIRASVGKMIDGIGR FYI Protein QMCTELKLSD HEGRLIQNS LTIEK MVLSAFDERRN KYLEEH PSAGKDPKKTGGPIY RRVDGKWMRELVLYDKEEIRRIWRQ ANNGEDATSGLTHI MIWHSNLND A TYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKG IGTMVMELIRMVKRGIN DRNFWRGENGRKTRSAYERMCNILKGKFQTAAQRAM V DQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACAYGP AVSSGYDFEKEGYSLVGIDPFKLLQNSQIYSLIRPNENPAHK SQLVWMACHSAAFEDLRLLSFIRGTKVSPRGKLSTRGVQIA SNEN MDNMGSSTLELRSG YWAIRTRSGGNTNQQ RASAGQTSVQPTFSVQRNLPFEKSTIMAAFTGNT EGRTSDMR AEIIR MMEGAKPEEVSFRGRGVFELSDEKATNPIVPSFDMSNEGSY FFGDNAEEYD N (underline = highly conserved;bold = hypervariable) 174 H3N2 NS1 MDSNTVSSFQVDCFLWHIRKQVVDQKLSDAPFLDRLRRDQ Protein RSLRGRGNTLGLDIKAATHVGKQIVEKILKEESDEALKMTMVSTPASRYITDMTIEELSRNWFMLMPKQKVEGPLCIRMDQAIMEKNIMLKANFNVIFGRLETIVLLRAFTEEGAIVGEISPL PS FPGHTIEDVKNAIGVLIGGLEWNDNTVRVSKNLQRFAWR SSNENGGPPLTPK (underline = highly conserved;bold = hypervariable) 175 H3N2 NS2 MDSNTVSSFQDIL LRMSKMQLGSSSEDLNGMITQFESLKI YR ProteinDSLGEAVMRMGDLHLLQNRNGKWREQLGQKFEEIRWLIEE VRHRLKT TENSFEQITFMQALQLLFEVEQEI RTFSFQLI (underline = highly conserved; bold = hypervariable)176 H3N2 PA MEDFVRQCFNPMIVELAEKAMKEYGEDL KIETNKFAAICTH ProteinLEVCFMYSDFHFINEQGESIVVELD DPNALLKHRFEIIEGRD RTMAWTVVNSICNTTGAGKPKFLPDLYDYKENRF I EIGVTR REVHIYYLEKANKIKSE N THIHIFSFTGEEMATKADYTLDEESRARIKTRLFTIRQEMANRGLWDSFRQSERGEETIEE KFEITG TMRRLADQSLPPNFS CLENFRAYVDGFEPNGCIEGKLSQMS KEV NAQIEPFLKTTPRPIKLPSGPPCY QRSKFLLMDALKLSIEDPSHEGEGIPLYDAIKC IKTFFGWKEP YIVKPHEKGINSNYLLSWKQVLSELQDIENEEKIPRTKNMKKTSQLKWALGENMAP EKVDFENCRDISDLKQYDSEEPELRSLSSWIQSEFNKACELTDSVWIELDEIGEDVAPIEHIASMRRNYFTAEVSHCRATEYIMKGVYINTALLNASCAAMDDFQLIPMISKCRTKEGRRKTNLYGFIIKGRSHLRNDTDVVNFVSMEFSLTDPRLEPHKWEKYCV LEIGDMLLR SAIGQISRPMFLYVRTNGTSK V KMKWGMEMR RCLLQSLQQIESMIEAESS VKEKDMTKEFFENKSEA WPIGESPKGVEEGSIGKVCRTLLAKSVFNSLYASPQLEGFSAESRKLL L IVQALRDKLEPGTFDLGGLYEAIEECLINDPWVLLNASWF NSFLTHALK(underline = highly conserved; bold = hypervariable) 177 H3N2 PA-XMEDFVRQCFNPMIVELAEKAMKEYGED L KIETNKFAAICTH Protein LEVCFMYSDFHFINEQGESIVVELD DPNALLKHRFEIIEGRD RTMAWTVVNSICNTTG AG KPKFLPDLYDYKENRF IEIGVTR REVHIYYLEKANKIKSE N THIHIFSFTGEEMATKADYTLDEESRARIKTRLFTIRQEMANRGLWDSFVSP KEAKKQLKKNLKS QELCAGLPTKVSHRTSPALRILEPMWMDSNRTAALRASFLK CPKK (underline = highly conserved;bold = hypervariable) 178 H3N2 PB1MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMD Protein TVNRTHQYSERGKWTTNTETGAPQLNPIDGPLPEDNEPSGY AQTDCVLEAMAFLEESHPGIFENSCLETMEAVQQTRVDKLT QGRQTYDWTLNRNQPAATALANTIEVFRSNGLTANESGRLIDFLKDVMESMDKEEMEITTHFQRKRRVRDNMTKKMVTQRTIGKKKQRVNKRGYLIRALTLNTMTKDAERGKLKRRAIATPGMQIRGFVYFVETLARSICEKLEQSGLPVGGNEKKAKLANVVRKMMTNSQDTELSFTITGDNTKWNENQNPRMFLAMITYITKNQPEWFRNILSIAPIMFSNKMARLGKGYMFESKRMKLRTQIPAEMLASIDLKYFNESTRKKIEKIRPLLIDGTASLSPGMM MGMFNMLSTVLGVSILNLGQKKYTKTTYWWDGLQSSDDF ALIVNAPNHEGIQAGVDRFYRTCKLVGINMSKKKSYIN K TGTFEFTSFFYRYGFVANFSMELPSFGVSGINESADMSIGVTVIKNNMINNDLGPATAQMALQLFIKDYRYTYRCHRGDTQIQTR RSFE IKKLWDQTQSRTGLLVSDGGPNLYNIRNLHIPEVCLK WELMD ENYR GRLCNPLNPFVSHKEIESVNN AVVMPAHGPAKSMEYDAVATTHSWIPKRNRSILNTSQRGILEDEQMYQKCC NLFEKFFPSSSYRRPIGISSMVEAMVSRARIDARIDFESGRIK KEEFSEIMKICSTIEELRRQK(underline = highly conserved; bold = hypervariable) 179 H3N2 PB2MERIKELR N LMSQSRTREILTKTTVDHMAIIKKYTSGRQEKN Protein P SLRMKWMMAMKYPITADKRITEMV PERNEQGQTLWSK MS DAGSDRVMVSPLAVTWWNRNGPVTSTVHYPKVYKTYF D KVERLKHGTFGPVHFRNQVKIRRRVD I NPGHADLSAKEAQDVIMEVVFPNEVGARILTSESQLTITKEKKEEL RDCKIS PL MVAYMLERELVRKTRFLPVAGGTSSIYIEVLHLTQGTCWEQ MYTPGG GVRNDDVDQSLIIAARNIVRRAAVSADPLASLLEM CHSTQIGGTRMVDILR QNPTEEQAVDICKAAMGLRISSSFSF GGFTFKRTSGSSVKKEEEVLTGNLQTLRIRVHEGYEEFTMV GK RATAILRKATRRL VQLIVSGRDE QSIAEAIIVAMVFSQEDCMIKAVRGDLNFVNRANQRLNPMHQLLRHFQKDAKVLFQ NWG VEHIDSVMGMVGVLPDMTPSTEMSMRGIRVSKMGVD EYSSTERVVVSIDRFLRVRDQRGNVLLSPEEVSETQGTER LTITYSSSMMWEINGPESVLVNTYQWIIRNWEAVKIQWSQNPA MLYNKMEFEPFQSLVPKATRSQYSGFVRTLFQQMRDVLGT FDT A QIIKLLPFAAAPP K QSRMQFSSLTVNVRGSG M RILVRGNSPVFNYNK TTKRLTILGKDAGTLIEDPDESTS GVESAVLRG FLIIGKEDRRYGPALSINELSNLAKGEKANVLIGQGDVVLV MKRKRDSSILTDSQTATKRIRMAIN(underline = highly conserved; bold = hypervariable 180 Influenza BMLEPFQILS ICSFILSALHFMAWTIGHLNQIKRGVNMKIRIKG bm2 Protein PNKETINREVSILRHSYQKEIQAKE AMKEVLSDNMEVLSDHI V IEGLSAEEIIKMGETVLEVEELH(underline = highly conserved; bold = hypervariable) 181 Influenza BMSLFGDTIAYLLSLTEDGEGKAELAEKLHCWFGGKEFDLDS bm1 ProteinALEWIKNKRCLTDIQKALIGASICFLKPKDQERKRRFITEPLS GMGTTATKKKGLILAERKMR KCVSFHEAFEIAEGHESSALL YCLMVMYLNPGNYSMQVKLGTLCALCEKQASHSHRAHSRAARSSVPGVRREMQMVSAMNTAKTMNGMGKGEDVQKLAEELQSNIGVLRSLGASQKNGEGIAKDVMEVLKQSSMGNSAL VKKYL(underline = highly conserved; bold = hypervariable) 182 Influenza BMADNMTTTQIEWRMKKMAIGSS IHSSSVLMKDIQSQFEQL nep ProteinKLRWESYPNLVKSTDYHQKRETIRLVTEELYLLSKRIDDNIL FHKTVIANSSIIADMVVSLSLLETLYEMKDVVEVYSRQCL (underline = highly conserved;bold = hypervariable) 183 Influenza B MADNMTTTQIEVGPGATNATINFEAGILECYERLSWQRALD ns1 ProteinYPGQDRLNRLKRKLESRIKTHNKSEPESKRMSLEERKAIGVKMMKVLLFMNPSAGIEGFEPYCMKSSSNSNCPKYNWTDYP STPGRCLDDIEEEPDDVDGPTEIVLRDMNNKDARQKIKEEV NTQKEGKFRLTIKRDMRNVLSLRVLVNGTFLKHPNGYKSLSTLHRLNAYDQSGRLVAKLVATDDLTVEDEEDGHRILNSLFE RLNEGHSKPIRAAETAVGVLSQFGQEHRLSPEEGDN (underline = highly conserved; bold = hypervariable)184 Influenza B MADNMTTTQIEVVRMKKMAIGSSTHSSSVLMKDIQSQFEQL ns2 ProteinKLRWESYPNLVKSTDYHQKRETIRLVTEELYLLSKRIDDNIL FHKTVIANSSIIADMVVSLSLLETLYEMKDVVEVYSRQCL (underline = highly conserved;bold = hypervariable) 185 Influenza BMDTFITRNFQTTIIQKAKNTMAEFSEDPELQPAMLFNICVHL paEVCYVISDMNFLDEEGKAYTALEGQGKEQNLRPQYEVIEG ProteinMPRTIAVVMVQRSLAQEHGIETPKYLADLFDYKTKRFIEVGITKGLADDYFWKKKEKLGNSMELMIFSYNQDYSLSNESSLDEEGKGRVLSRLTELQAELSLKNLWQVLIGEEDVEKGIDFKLGQTISRLRDISVPAGFSNFEGMRSYIDNIDPKGAIERNLARMS PLVSVTPKKLK WEDLRPIGPHIYNHELPEVPYNAFLLMSDEL GLANMTEGKSKKPKTLAKECLEKYSTLRDQTDPILI M KSEKANENFLWKLWRDCVNTISNEE M SNELQKTNYAKWATGDGLTYQKIMKEVAIDDETMCQEEPKIPNKCRVAAWVQTEMNL LSTLTSKRALDLPEIGPD VAPVEHVGSERRKYFVNEINYCKA STVMMKYVLFHTSLLNESNASMGKYKVIPITNR V VNEKGESFDMLYGLAVKGQSHLRGDTDVVTVVTFEFSSTDPRVDS GK WPKYTVFRIGSLFV SGREKSVYLYCRVNGTNKIQMKWGME ARRCLLQSMQQMEAIVEQESSIQGYDMTKACFKGDRVNSPKTFSIGTQEGKLVKGSFGKALRVIFTKCLMHYVFGNAQLEGFSAESRRLLLLIQALKDRKGPWVFDLEGMYSGIEECISNNPWVIQSAYWFNEWLGFEKEGSKVLESVDEIMDE (underline = highly conserved;bold = hypervariable) 186 Influenza B MNINPYFLFIDVP IQAAISTTFPYTGVPPYSHGTGTG Y TIDTVI pb1 Protein RTHEYSNKGKQYVSDITGCTMVDPTNGPLPEDNEPSAYAQL DCVLEALDRMDEEHPGLFQAASQNAMEALMVTTVDKLTQGRQTFDWTVCRNQPAATALNTTITSFRLNDLNGADKGGLV PFCQDIIDSLD KPEMTFFSVKNIKKKLPAKNRKGFLIKRIPMK VKDRISRVEYIKRALSLNTMTKDAERGKLKRRAIATAGIQIRGFVLVVENLAKNICENLEQSGLPVGGNEKKAKLSNAVAKMLSNCPPGGISMTVTGDNTKWNECLNPRIFLAMTERITRDSP I WFRDFCSIAPVLFSNKIARLGKGFM ITSKTKRLKAQIPCPDLF SIPLERYNEETRAKL KK LKPFFNEEGTASLSPGMMMGMFNMLSTVLGVAALGIKNIGNKEYLWDGLQSSDDFALFVNAKDEETCMEGINDFYRTCKLLGINMSKKKSYCNETGMFEFTSMFYRDGFVSNFAMEIPSFGVAGVNESADMAIGMTIIKNNMINNGMGPATAQTAIQLFIADYRYTYKCHRGDSKVEGKRMKIIKELWENTKGRDGLLVADGGPNIYNLRNLHIPEIVLKYNLMDPEYKGRLLHPQNPFVGHLSIEGIKEADITPAHGPVKKMDYDAVSGTHSWRTKRNRSILNTDQRNMIEEEQCYAKCCNLFEACFNSASYRKPVGQHSMLEAMAHRLRMDARLDYESGRMSKDDF EKAMAHLGEIGYT(underline = highly conserved; bold = hypervariable) 187 Influenza BMTLAKIELLKQLLRDNEAKTVLKQTTVDQYNIIRKFNTSRIE pb2 ProteinKNPSLRMKWAMCSNFPLALTKGDMANRIPLEYKGIQLKTNAEDIGTKGQMCSIAAVTWWNTYGPIGDTEGFEK VYESFFLRKMRLDNATWGRITFGPVERVRKRVLLNPLTKEMPPDEASNVIMEILFPKEAGIPRESTWIHRELIKEKREKLKGTMITPIVLAYMLERELVARRRFLPVAGATSAEFIEMLHCLQGENWRQIYHPGGNKLTESRSQSMIVACRKIIRRSIVASNPLELAVEIANKTVI DTEPLKSCL TAIDGGDVACDIIRAALGLKIRQRQRFGRLELK RISGRGFKNDEEILIGNGTI QKIGIWDGEEEFHVRCGECRGIL KKSKM RM EKLLINSAKKEDM K DLIILCMVFSQDTRMFQGVRGEINFLNRAGQLLSPMYQLQRYFL S RSNDLFDQWGYEESP KASELHGINE L MNASDYTLKGVVVTK NVIDDFSSTETEKVS ITKNLSLIKRTGEVIMGANDVSELESQAQLMITYDTPKMWEMGTTKELVQNTYQWV L KNLVTEKAQFLLGKEDMFQWDAFEAFESIIPQKMAGQYSGFARAVLKQMRDQEVMKTDQFIKLLPFCFSPPKLRSNGEPYQFLRLVLKGGGENFIEVRKGSPLFSYNPQTEVLTICGRMMSLKGKIEDEERNRSMGNAVLAGFLVSGKYDPDLGDFKTIEELEKLKPGEKANILLYQGKPVKVVKRKR YSALSNDISQGIKRQRMTVESMGWALS(underline = highly conserved; bold = hypervariable) 188 H1N1 MMSLLTEVETYVLSI I PSGPLKAEIAQRLE S VFAGKNTDLEAL ProteinMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRF I QNAL NGNGDPNNMDRAVKLYKKLKREITFHGAKE VSLSYST GAL ASCMGLIYNRMG TVTTEAAFGLV CATCEQIADSQH RSHRQ MATTTNPLIRHENRMVLASTTAKAMEQ V AGSSEQAAEAME VA NQTRQMVHAMRT IGTHPSSSAGLRDDLLENLQAYQKR MGVQMQRFK (underline = highly conserved;bold = hypervariable) 189 H1N1 ns1MDSNTMSSFQVDCFLWHIRKRFADNGLGDAPFLDRLRRDQ ProteinKSLKGRGNTLGLDIETATLVGKQIVEWILKEESSETLRMTIASVPTSRYLSDMTLEEMSRDWFMLMPRQKIIGPLCVRLDQAIMEKNIVLKANFSVIFNRLETLILLRAFTEEGAIVGEISPLPSLPGHTYEDVKNAVGVLIGGLEWNGNTVRVSENIQRFA WRNCDENGRPSLPPEQK(underline = highly conserved; bold = hypervariable) 190 H1N1 paMEGFVRQCFNPMIVELAEKAMKEYGEDPKIETNKFAAICTH ProteinLEVCFMYSDFHFIDERGESIIVESGDPNALLKHRFEIIEGRDR IMAWTVVNSICNTTGVEKPKFLPDLYDYKENRFIEIGVTRREVHIYYLEKANKIKSEKTHIHIFSFTGEEMATKADYTLDEESRARIKTRLFTIRQEMASRSLWDSFRQSERGEETIEEKFEITGTMRKLADQSLPPNFSSLENFRAYVDGFEPNGC IEGKLSQMSKEVNAKIEPFLRTTPRPLRLPDGPLCHQRSKFLLMDALKLSIED PS HEGEGIPLYDAIKCMK TFFGWKEPNIVKPHEKGINPNYL MTWKQVLAELQDIENEEKIPRTKNMKRTSQLKWALGENMAPEKVDFDDCKDVGDLKQYDSDEPEPRSLASWVQN EFNKACELTDSSWIELDEIGEDVAPIEHIASMRRNYFTAEVSHCRATEYIMKGVYINTALLNASCAAMDDFQLIPMISKCRTKEGRRKTNLYGFIIKGRSHLRNDTDVVNFVSMEFSLTDPRLEPHKWEKYCVLEIGDMLLRTAIGQVSRPMFLYVRTNGTSKIKMKWGMEMRRCLLQSLQQIESMIEAESSVKEKDMTKEFFENKSETWPIGESPRGVEEGSIGKVCRTLLAKSVFNSLYASPQLEGFSAESRKLLLIVQALRDNLEPGTFDLGGLYEAIEECLINDPWVLLNA SWFNSFLTHALK(underline = highly conserved; bold = hypervariable) 191 H1N1 pb1MDVNPTLLFLKIPAQNAISTTFPYTGDPPYSHGTGTGYTMDT ProteinVNRTHQYSEKGKWTTNTETGAPQLNPIDGPLPEDNEPSGYAQTDCVLEAMAFLEESHPGIFENSCLETMEVVQQTRVDKLTQGRQTYDWTLNRNQPAATALANTIEVFRSNGLTANESGRLIDFLKDVMESMNKEEIEITTHFQRKRRVRDNMTKKMVTQRTIGKKKQRLNKRGYLIRALTLNTMTKDAHRGKLKRRAIATPGMQIRGFVYFVETLARSICEKLEQSGLPVGGNEKKAKLANVVRKMMTNSQDTEISFTITGDNTKWNENQNPRMFLAMITYITRNQPEWFRNILSMAPIMFSNKMARLGRGYMFESKRMKIRTQIPAEMLASIDLKYFNESTKKKIEKIRPLLIDGTASLSPGMMMGMFNMLSTVLGVSILNLGQKKYTKTIYWWDGLQSSDDFALIVNAPNHEGIQAGVDRFYRTCKLVGINMSKKKSYINKTGTFEFTSFFYRYGFVANFSMELPSFGVSGVNESADMSIGVTVIKNNMINNDLGPATAQMALQLFIKDYRYTYRCHRGDTQIQTRRSFELKKLWDQTQSKVGLLVSDGGPNLYNIRNLHIPEVCLKWELMDDDYRGRLCNPLNPFVSHKEIDSVNNAVVMPAHGPAKSM EYDAVATTHSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEKFFPSSSYRRPVGISSMVEAMVSRARIDARVDFESGRIKKEEFSEIMKICSTIEELRRQK (underline = highly conserved;bold = hypervariable) 192 H1N1 pb2AMGLRISSSFSFGGFTFKRTSGSSVKREEEVLTGNLQTLKLT ProteinVHEGYEEFTMVGKRATAILRKATRRLIQLIVSGRDEQSIVEAIVVAMVFSQEDCMVKAVRGDLNFVNRANQRLNPMHQLLRHFQKDAKVLFLNWGVEPIDNVMGMIGILPDMTPSTEMSMRGVRVSKMGVDEYSNAERVVVSIDRFLRVRDQRGNVLLSPEEVSETQGTEKLTITYSSSMMWEINGPESVLINTYQWIIRNWETVKIQWSQNPTMLYNKMEFEPFQSLVPKAIRGQYSGFVRTLFQQMRDVLGTFDTTQIIKLLPFAAAPPKQSRMQFSSLTVNVRGSGMKILVRGNSPVFNYNKTTKRLTVLGKDAGTLTEDPDEGTAGVESAVLRGFLILGKEDRRYGPALSINELSNLAKGEKANVLIGQGDVVLVMKRKRDSSILTDSQTATKRIRMAIN (underline = highly conserved;bold = hypervariable) 193 H1N1 NS2MDSNTMSSFQDILMRMSKMQLGSSSEDLNGMVTRFESLKI ProteinYRDSLGETVMRMGDLHYLQSRNEKWREQLGQKFEEIRWLIEEMRHRLKATENSFEQITFMQALQLLLEVEQEIRAFSFQLI (underline = highly conserved;bold = hypervariable)

The invention claimed is:
 1. An immunogenic composition comprising oneor more polypeptides comprising an amino acid sequence at least 80%identical to the amino acid sequence of SEQ ID NO:1, wherein the one ormore polypeptides each comprise mutations to alanine or glycine at eachof residues 159, 415, 419, 490, and 491 relative to the amino acidsequence of SEQ ID NO:1.
 2. The immunogenic composition of claim 1,wherein the one or more polypeptides comprise an amino acid sequence atleast 90% identical to the amino acid sequence of SEQ ID NO:1.
 3. Theimmunogenic composition of claim 1, wherein the one or more polypeptidescomprise an amino acid sequence at least 95% identical to the amino acidsequence of SEQ ID NO:1.
 4. The immunogenic composition of claim 1,wherein the one or more polypeptides further comprise a mutation toalanine or glycine at one or more residue selected from the groupconsisting of 53, 114, 220, 275, and 277 relative to SEQ ID NO:1.
 5. Theimmunogenic composition of claim 4, wherein the one or more polypeptidescomprise a mutation to alanine or glycine at each residue selected fromthe group consisting of 53, 114, 220, 275, and 277 relative to SEQ IDNO:1.
 6. The immunogenic composition of claim 4, wherein the one or morepolypeptides further comprise a mutation to alanine or glycine at one ormore residue selected from the group consisting of 147, 180, 225, 228,300, and 319 relative to SEQ ID NO:1.
 7. The immunogenic composition ofclaim 6, wherein the one or more polypeptides comprise a mutation toalanine or glycine at each residue selected from the group consisting of147, 180, 225, 228, 300, and 319 relative to SEQ ID NO:1.
 8. Theimmunogenic composition of claim 1, wherein the one or more polypeptideseach comprise mutations to alanine at each of residues 159, 415, 419,490, and 491 relative to the amino acid sequence of SEQ ID NO:1.
 9. Theimmunogenic composition of claim 2, wherein the one or more polypeptideseach comprise mutations to alanine at each of residues 159, 415, 419,490, and 491 relative to the amino acid sequence of SEQ ID NO:1.
 10. Theimmunogenic composition of claim 3, wherein the one or more polypeptideseach comprise mutations to alanine at each of residues 159, 415, 419,490, and 491 relative to the amino acid sequence of SEQ ID NO:1.
 11. Theimmunogenic composition of claim 1, wherein the one or more polypeptidesfurther comprise a mutation to alanine at one or more residue selectedfrom the group consisting of 53, 114, 220, 275, and 277 relative to SEQID NO:1.
 12. The immunogenic composition of claim 1, wherein the one ormore polypeptides further comprise a mutation to alanine at each residueselected from the group consisting of 53, 114, 220, 275, and 277relative to SEQ ID NO:1.
 13. The immunogenic composition of claim 1,wherein the one or more polypeptides further comprise a mutation toalanine at one or more residue selected from the group consisting of147, 180, 225, 228, 300, and 319 relative to SEQ ID NO:1.
 14. Theimmunogenic composition of claim 1, wherein the one or more polypeptidesfurther comprise a mutation to alanine at each residue selected from thegroup consisting of 147, 180, 225, 228, 300, and 319 relative to SEQ IDNO:1.
 15. A method for immunizing a subject against infection with aninfluenza virus, inducing an immune response against influenza virus, orreducing an influenza virus infection in a subject in need thereof,comprising administering the immunogenic composition of claim
 1. 16. Amethod for immunizing a subject against infection with an influenzavirus, inducing an immune response against influenza virus, or reducingan influenza virus infection in a subject in need thereof, comprisingadministering the immunogenic composition of claim
 2. 17. A method forimmunizing a subject against infection with an influenza virus, inducingan immune response against influenza virus, or reducing an influenzavirus infection in a subject in need thereof, comprising administeringthe immunogenic composition of claim
 3. 18. A method for immunizing asubject against infection with an influenza virus, inducing an immuneresponse against influenza virus, or reducing an influenza virusinfection in a subject in need thereof, comprising administering theimmunogenic composition of claim 4.